HK1222183B - Elucidation of ion exchange chromatography input optimization - Google Patents

Description

Elucidating ion exchange chromatography input optimization

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/845,890, filed July 12, 2013, the disclosure of which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention provides methods for analyzing protein charge variants in polypeptide preparations using ionic strength gradient ion exchange chromatography.

Background of the Invention

In aqueous environments, proteins such as monoclonal antibodies (mAbs) have a large number of charged and polar amino acids on their surfaces (Barlow, DJ and Thornton, JM (1986) Biopolymers 25:1717). As the molecules interact with solution components, surface residues can undergo a variety of chemical and enzymatic modifications, resulting in a heterogeneous mixture of protein variants with slightly different surface electrostatics (Dick, LW et al., (2009) J. Chromatogr. B 877:3841; Liu, HW et al., (2008) Rapid Commun. Mass Spectrom. 22:4081; Miller, AK et al., (2011) J. Pharm. Sci. 100:2543; Wang, WR et al., (2011) Mol. Immunol. 48:860). According to a recent review by Vlasak, J and Ionescu, R (2008 Curr. Pharm. Biotechnol. 9: 468), cation exchange chromatography (CEC) is considered the gold standard for profiling the charge heterogeneity of protein therapeutics. Regulatory agencies generally require this charge-sensitive separation method to ensure production consistency during manufacturing and monitor the degradation level of protein therapeutics (Miller, AK, et al., (2011) J. Pharm. Sci. 100: 2543; He, XPZ (2009) Electrophoresis 30: 714; Sosic, Z et al., (2008) Electrophoresis 29: 4368; Kim, J et al., (2010) J. Chromatogr. B 878: 1973; Teshima, G et al., (2010) J. Chromatogr. A 1218: 2091).

Ion exchange chromatography (IEC) is generally performed in a bind and elute mode. Typically, a protein sample (such as a mAb) is introduced into the stationary phase under conditions that promote protein binding to the column (i.e., in 100% buffer A). A salt or pH gradient (i.e., increasing % buffer B) is applied to induce the elution of different charged proteins in sequence. IEC methods are generally product-specific. Developing robust methods that can withstand temperature and pH fluctuations and fully address charge heterogeneity is resource-intensive. There is a need to develop methods for optimizing buffer systems that allow for the formation of robust assays to determine the presence of contaminants in a variety of polypeptide products. The present invention provides methods for predicting optimal ion exchange conditions based on mathematical models of polypeptides and buffer systems.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

Summary of the Invention

In some aspects, the present invention provides a method for determining optimal ion exchange chromatography separation conditions for analyzing a plurality of compositions, wherein each composition comprises a polypeptide together with one or more contaminants, the method comprising a) plotting a net charge versus pH curve at a selected temperature based on the amino acid composition of the polypeptides of the two or more compositions, and b) determining the inflection point of the net charge versus pH curve at or near neutral pH by determining the second derivative of the curve of step a); wherein the optimal ion exchange chromatography separation condition is a pH at approximately a common inflection point for the polypeptides of the one or more compositions. In some embodiments, the method further comprises c) determining the change in pH of the inflection point of the net charge versus pH curve as a function of temperature (dIP/dT) for the polypeptides of the two or more compositions, and d) selecting a buffer for chromatography, wherein the change in acid dissociation constant of the buffer as a function of temperature (dpKa/dT) is substantially the same as the dIP/dT of the polypeptide.

In other aspects, the present invention provides a method for determining optimal ion exchange chromatography separation conditions for analyzing a composition comprising a polypeptide together with one or more contaminants, the method comprising a) plotting a net charge versus pH curve at a selected temperature based on the amino acid composition of the polypeptide, and b) determining the inflection point of the net charge versus pH curve at or near neutral pH by determining the second derivative of the curve of step a); wherein the optimal ion exchange chromatography separation conditions are at a pH at about the common inflection point of the polypeptide.

In some embodiments, if the net charge at the inflection point is positive, a cation exchange material is used for ion exchange chromatography. In some embodiments, the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material. In other embodiments, if the net charge at the inflection point is negative, an anion exchange material is used for chromatography. In some embodiments, the anion exchange chromatography material is a quaternary amine chromatography material or a tertiary amine chromatography material. In yet other embodiments, a mixed mode chromatography material is used for chromatography. In some embodiments, the mixed mode ion exchange material is a mixture of a sulfonated chromatography material or a carboxylated chromatography material and a quaternary amine chromatography material or a tertiary amine chromatography material, which are loaded sequentially.

In some embodiments, the buffer provides effective buffering capacity at the inflection point pH. In some embodiments, the dpKa/dT of one or more polypeptides of the composition is approximately -0.02 pH units. In some embodiments, the temperature change is from about 20°C to about 70°C. In other embodiments, the temperature change is from about 20°C to about 50°C. In some embodiments, dpKa/dT = dpKa/dT ± 50%. In some embodiments, the net charge of the polypeptide in the buffer selected in step d) varies by less than 0.5 above 30°C. In some embodiments, the buffer selected in step d) is used in the chromatography at a concentration ranging from about 5 mM to about 250 mM.

In some embodiments of the above embodiments, the buffer composition further comprises a salt. In other embodiments, the salt is NaCl, KCl, (NH ₄ ) ₂ SO ₄ or Na ₂ SO _4. In some embodiments, the concentration of the salt is from about 1 mM to about 1M.

In some embodiments of the methods of the present invention, the polypeptide is an antibody or immunoadhesin or a fragment thereof. In some embodiments, the polypeptide is a monoclonal antibody or a fragment thereof. In some embodiments, the antibody is a human antibody. In other embodiments, the antibody is a humanized antibody. In yet other embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is an antibody fragment.

In some embodiments of the methods of the present invention, the contaminant is a variant of the polypeptide. In some embodiments, the contaminant is a degradation product of the polypeptide. In some embodiments, the contaminant is a charge variant of the polypeptide.

In some aspects, the present invention provides a method for analyzing a composition, wherein the composition comprises a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) determining optimal pH and temperature conditions for ion exchange separation of a plurality of compositions according to the methods of the present invention, each composition comprising a target polypeptide and one or more contaminants, b) binding the polypeptide and one or more contaminants from the composition to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffer determined by the methods of the present invention; c) eluting the polypeptide and one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises the buffer and a salt, wherein the concentration of the salt increases in the gradient over time, wherein the polypeptide and the one or more contaminants are separated by the gradient; and d) detecting the polypeptide and the one or more contaminants.

In some aspects, the present invention provides a method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) binding the polypeptide and the one or more contaminants to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffering agent, and wherein the pH and temperature of the chromatography have been optimized for a plurality of target polypeptides by: i) plotting a net charge versus pH curve at a selected temperature, wherein the curve is based on the amino acid composition of the polypeptides of two or more target polypeptides, and ii) determining the inflection point of the net charge versus pH curve by determining the second derivative of the curve of step i); wherein the optimal ion exchange chromatography condition is a pH at a common inflection point for the two or more target polypeptides; b) eluting the polypeptide and the one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises a buffering agent and a salt, wherein the polypeptide and the one or more contaminants are separated by the gradient; and c) detecting the polypeptide and the one or more contaminants. In some embodiments, the selected temperature is ambient temperature. In some embodiments, a buffer is determined by: a) determining the change in the pH of the inflection point of the net charge versus pH curve as a function of temperature (dlP/dT) for two or more target polypeptides, and b) selecting a buffer having an acid dissociation constant as a function of temperature (dpKa/dT) that is substantially the same as the dlP/dT for one or more target polypeptides having a common inflection point. In some embodiments, the buffer provides an effective buffering capacity at the inflection point pH.

In some aspects, the present invention provides a method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) binding the polypeptide and one or more contaminants to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffering agent, and wherein the pH and temperature of the chromatography have been optimized for a plurality of target polypeptides; b) eluting the polypeptide and one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises a buffering agent and a salt, wherein the polypeptide and the one or more contaminants are separated by the gradient; and c) detecting the polypeptide and the one or more contaminants. In some embodiments, the buffer is N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES) or (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In other embodiments, the concentration of the buffer is from about 5 mM to about 20 mM. In some embodiments, the temperature change is from about 20°C to about 70°C. In other embodiments, the temperature change is from about 20°C to about 50°C. In some embodiments, dpKa/dT = dIP/dT ± 50%. In some embodiments, the net charge of the polypeptide in the buffer varies by less than 0.5 above 30°C. In some embodiments, the buffer is used in chromatography at a concentration ranging from about 5 mM to about 250 mM.

In some embodiments of the above embodiments, the buffer composition further comprises a salt. In other embodiments, the salt is NaCl, KCl, (NH ₄ ) ₂ SO ₄ or Na ₂ SO _4. In some embodiments, the concentration of the salt is from about 1 mM to about 1 M. In some embodiments, the salt concentration increases from about 0 mM to about 100 mM in about 100 minutes. In other embodiments, the salt concentration increases from about 0 mM to about 80 mM in about 40 minutes.

In some embodiments of the methods of the present invention, the polypeptide is an antibody or immunoadhesin or a fragment thereof. In some embodiments, the polypeptide is a monoclonal antibody or a fragment thereof. In some embodiments, the antibody is a human antibody. In other embodiments, the antibody is a humanized antibody. In yet other embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is an antibody fragment.

In some embodiments of the methods of the present invention, the contaminant is a variant of the polypeptide. In some embodiments, the contaminant is a degradation product of the polypeptide. In some embodiments, the contaminant is a charge variant of the polypeptide.

In some embodiments, the chromatography material is a cation exchange chromatography material. In other embodiments, the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material.

In some aspects, the present invention provides a method for analyzing a plurality of polypeptide compositions, wherein each polypeptide composition comprises a polypeptide and one or more charge variants of the polypeptide, wherein the method effectively separates the polypeptide from its charge variants; for each polypeptide composition, the method comprises, a) binding the polypeptide and one or more charge variants to an ion exchange chromatography material using a loading buffer at a flow rate of about 1 mL/minute, wherein the loading buffer comprises 10 mM HEPES buffer at about pH 7.6 at about 40° C.; b) eluting the polypeptide and charge variant contaminants from the ion exchange chromatography material using a gradient of elution buffer, wherein the elution buffer comprises 10 mM HEPES buffer at about pH 7.6 and NaCl, wherein the concentration of NaCl in the gradient increases from about 0 mM to about 80 mM over about 40 minutes, wherein the polypeptide and its charge variants are separated by the gradient; and c) detecting the polypeptide and one or more charge variants. In some embodiments, the plurality of polypeptide compositions comprises different polypeptides. In some embodiments, the plurality of polypeptide compositions comprises polypeptides with different pIs. In some embodiments, the polypeptide composition is an antibody composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a plot of the calculated net charge versus pH for the monoclonal antibody mAb1 (solid blank line) and its two charge variants. The dashed lines represent the acidic variants with two negative charges and the basic variants with two positive charges as shown. The curves were generated using the amino acid sequence composition of mAb1 and its variants. The asterisk indicates the inflection point of the curve. A platform IEC method performed at the inflection point pH will provide optimal resolution and robustness relative to pH.

FIG2A shows a plot of the percentage of protonated histidine (positively charged) in a population (eg, a polypeptide solution) on a pH scale.

Figure 2B shows the number of deprotonated histidines (charge frequency) in a polypeptide containing 10 histidine residues at pH 6.5 and pH 7.5. It shows that at the inflection point (pH 7.5), most of the histidine residues are deprotonated and uncharged.

Figure 2C shows an example of four polypeptide molecules each having two histidine residues. At the pKa of His (pH 6.0), 50% of the His residues are protonated and 50% are deprotonated. The charge state combinations of the His residues on these four molecules are binomially distributed at the pKa: one molecule has both His residues protonated; two molecules have one His residue protonated and the other His residue deprotonated; and one molecule has both His residues deprotonated.

Figure 3 is a graph of common monoclonal antibodies in terms of charge frequency of polar amino acids as a function of pH. The probabilities of the most common charge states of the six amino acids contributing to the calculated charge at 37°C at different pH are plotted as solid lines, and the weighted combination of these amino acid residues for mAb1 is plotted as dashed lines.

Figure 4 shows the Shannon entropy of mAb1 at different pH values at 22° C. Table 3 lists the types and numbers of amino acid residues that contributed to the net charge calculation.

Figure 5 shows a 3D visualization of the charge distribution and charge distribution frequency within the mAb1 population at various pH values at 37°C. The charge distribution is most uniform at the inflection point of the net charge vs. pH curve, with a frequency of approximately 0.7. At pH values further from the inflection point, the charge distribution is broader, with a frequency of 0.15. Because IEC separation is based on charge, higher charge distribution frequencies result in narrower peaks and improved resolution.

Figure 6 is a 2D representation of Figure 5, i.e., a net charge versus pH curve of mAb 1. When the temperature is 37°C, the inflection point is at pH 7.5.

Figure 7 is a graph showing the net charge of various mAb products as a function of pH at 37°C. The calculated net charge for each mAb is based on the mAb's amino acid sequence. Some mAbs have different framework amino acid sequences. At 37°C, the inflection point for all curves is approximately pH 7.5.

Figure 8 is a graph showing the calculated inflection points for a number of mAb products at 22°C (diamonds), 37°C (triangles), and 50°C (squares).

FIG9 is a graph showing the net charge of mAb2 versus pH at temperatures ranging from 22°C to 50°C.

Figure 10A shows the rate of change of the inflection point. Figure 10B shows the change of dIP as a function of temperature for selected mAbs. The rate of change is almost the same.

FIG11 is a graph showing the change in net charge of mAb2 in given buffers (phosphate, HEPES, ACES, and Tris) as a function of temperature.

Figure 12 shows an overlaid chromatogram of numerous mAbs with varying pIs using the same chromatography method. Buffer A was 5 mM ACES pH 7.5 at 37°C. Buffer B was 180 mM NaCl in buffer A. The salt gradient was 0 mM NaCl to 100 mM NaCl over 100 minutes at 37°C, or 1 mM/minute. The flow rate was 0.8 mL/minute. The column was a MabPac SCX-10 column (4 x 250 mm).

Figure 13 shows the robustness of IEC of mAb 4 as a function of pH. Chromatographic conditions were as in Figure 12, except that the gradient was 1.5 mM NaCl/min.

Figure 14 shows the robustness of IECs of three mAbs as a function of temperature. The chromatography conditions were identical for all three antibodies and as described for Figure 13 .

Figure 15 shows the robustness of IECs for three mAbs as a function of temperature. The chromatography conditions were identical for all three antibodies. The chromatography conditions were as in Figure 13, except that the buffer was 10 mM HEPES.

Figure 16 shows a comparison of IEX chromatography curves for three mAbs using a multi-product approach and using a method developed for each mAb. The multi-product approach was 5 mM ACES pH 7.5 at 37°C with a gradient from 0 mM NaCl to 75 mM NaCl (1.5 mM/min) over 50 minutes and a flow rate of 0.8 mL/min. The buffer and temperature used for the product-specific approaches were different. For mAb8, it was 20 mM MES pH 6.5 at 30°C; for mAb25, it was 20 mM HEPES pH 7.6 at 42°C; and for mAb26, it was 20 mM ACES pH 7.1 at 40°C. The column was a MabPac SCX-10 column (4×250 mm).

Figure 17 shows the use of multi-product chromatography conditions using mAb8 on four different columns: ProPac WCX-10 (10 μm, 4×250 mm), YMC (5 μm, 4×100 mm), AntiBodix (5 μm, 4×250 mm), and MabPac SCX-10 (10 μm, 4×250 mm). The chromatography conditions were the same as those described for Figure 13. The inset shows the expansion of the variant peaks.

Figure 18 shows the use of multi-product chromatography conditions for mAb 8 on ProPac WCX-10 columns of varying dimensions: 4 × 250 mm, 4 × 100 mm, and 4 × 50 mm. The shorter columns resulted in shorter run times. The chromatograms are normalized to the main peak. The chromatographic conditions were the same as those described for Figure 15 , with the exception of the gradient time.

Figure 19 shows the relative % curve of the main peak for the most robust DOE study of GMP.

Figure 20 shows the relative % curve of the main peak for the most robust DOE study of GMP.

Figure 21 shows the relative % curve of the main peak for the most robust DOE study of GMP.

Detailed Description of the Invention

The present invention provides a method for determining optimal ion exchange chromatography separation conditions to analyze a composition comprising a polypeptide together with one or more pollutants, the method comprising a) drawing a net charge versus pH curve at a selected temperature based on the amino acid composition of the polypeptide, and b) determining the inflection point (IP) of the net charge versus pH curve at or near neutral pH by determining the second derivative of the curve of step a); wherein the optimal ion exchange chromatography separation condition is the pH at approximately the common inflection point of the polypeptide. In some embodiments, the charge distribution frequency is determined by calculating the Shannon entropy of the polypeptide at different pH values at a given temperature. As the Shannon entropy decreases, the charge distribution of the polypeptide in the composition becomes more uniform. Therefore, the ability to distinguish between a polypeptide and its charge variants is improved.

In some embodiments, the present invention provides methods for determining a buffer for optimal ion exchange chromatography separation conditions for analyzing a composition comprising a polypeptide together with one or more contaminants. In some embodiments, the buffer is selected such that the change in the acid dissociation constant with temperature (dpKa/dT) is approximately equal to the change in the inflection point with temperature (dIP/dT) as described above.

In some aspects, the present invention provides a method for determining optimal ion exchange chromatography separation conditions for analyzing a plurality of compositions, wherein each composition comprises a polypeptide together with one or more contaminants, the method comprising a) plotting a net charge versus pH curve at a selected temperature based on the amino acid composition of the polypeptides of the two or more compositions, and b) determining the inflection point of the net charge versus pH curve at or near neutral pH by determining the second derivative of the curve of step a); wherein the optimal ion exchange chromatography separation condition is a pH at approximately a common inflection point of the polypeptides of the one or more compositions. Thus, the method can be used to analyze a plurality of products without the need to develop a dedicated protocol for each product.

I. Definition

The terms "polypeptide" or "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin). Also included within the scope of this definition are, for example, polypeptides that contain one or more analogs of an amino acid (e.g., containing non-natural amino acids, etc.) and other modifications known in the art. As used herein, the terms "polypeptide" and "protein" particularly encompass antibodies.

As used herein, the term "polypeptide charge variant" refers to a polypeptide that has been modified from its native state such that the charge of the polypeptide is altered. In some examples, the charge variant is more acidic than the parent polypeptide; that is, it has a lower pI than the parent polypeptide. In other examples, the charge variant is more basic than the parent polypeptide; that is, it has a higher pI than the parent polypeptide. Such modifications can be engineered or the result of natural processes such as oxidation, deamidation, C-terminal processing of lysine residues, N-terminal pyroglutamate formation, and glycation. In some examples, the polypeptide charge variant is a glycoprotein in which the glycans attached to the protein are modified such that the charge of the glycoprotein is altered compared to the parent glycoprotein, for example, by the addition of sialic acid or its derivatives. As used herein, the term "antibody charge variant" refers to an antibody or fragment thereof that has been modified from its native state such that the charge of the antibody or fragment thereof is altered.

By "purified" polypeptide (e.g., antibody or immunoadhesin) is meant a polypeptide whose purity has been increased so that the polypeptide exists in a purer form than it does in its natural environment and/or when initially synthesized and/or amplified under laboratory conditions. "Purity" is a relative term and does not necessarily mean absolute purity.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or counteracts the biological activity of a native polypeptide. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native polypeptide. Suitable agonist or antagonist molecules include, in particular, agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of a native polypeptide, and the like. Methods for determining whether a polypeptide is an agonist or antagonist can comprise contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide.

A polypeptide that "binds" an antigen of interest, such as a tumor-associated polypeptide antigen target, is a polypeptide that binds to the antigen with sufficient affinity so that the polypeptide can be used as a diagnostic and/or therapeutic agent in target cells or tissues expressing the antigen, and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to "non-target" polypeptides will be less than about 10% of the binding of the polypeptide to its specific target polypeptide, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

With respect to binding of a polypeptide to a target molecule, the term "specific binding" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably distinguishable from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a structurally similar molecule that has no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, specific binding is demonstrated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

Antibodies are naturally occurring immunoglobulin molecules that have a variable structure that is all based on the immunoglobulin fold. For example, an IgG antibody has two heavy chains and two light chains that are bound by disulfide bonds to form a functional antibody. Each heavy and light chain itself comprises a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and provides function when interacting with immune effectors that are not antigen-specific. The antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a specific antigen.

The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. Variability is not evenly distributed across the 110 amino acids that comprise the variable domain. In contrast, the V region consists of relatively unchanging segments called framework regions (FRs) of 15-30 amino acids, separated by shorter regions of 9-12 amino acids each called "hypervariable regions" (HVRs) with extreme variability. The variable domains of native heavy and light chains each include four FRs connected by three hypervariable regions in a generally β-sheet configuration, the FRs forming a loop that connects the β-sheet structure and, in some cases, the portion that forms the β-sheet structure. The hypervariable regions in each chain are held together closely by the FRs and, together with the hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant structures are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as enabling the antibody to participate in antibody-dependent cellular cytotoxicity (ADCC).

Each V region generally comprises three HVRs, e.g., complementarity determining regions ("CDRs," each containing a "hypervariable loop"), and four framework regions. Thus, an antibody binding site (the minimum structural unit required to bind to a desired specific antigen with great affinity) will generally include three CDRs, and at least three, preferably four, framework regions interspersed therebetween to anchor and display the CDRs in an appropriate conformation. Classical four-chain antibodies have an antigen binding site defined by the cooperation of the _VH and _VL domains. Certain antibodies, such as camelid and shark antibodies, lack light chains and rely on a binding site formed solely by heavy chains. Single-domain engineered immunoglobulins can be prepared in which the binding site is formed solely by heavy or light chains, without cooperation between _VH and _VL .

The term "variable" refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed across the variable domains of an antibody. It is concentrated in three segments (called hypervariable regions) of the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each include four FRs that generally adopt a beta-pleated configuration connected by three hypervariable regions, which form loops that connect the beta-pleated structure and, in some cases, form portions of the beta-pleated structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant structures are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as enabling the antibody to participate in antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" (HVR) refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region may comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues about positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) in _VL and residues about positions 31-35B (H1), 50-65 (H2), and 95-102 (H3) in _VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in _{VL and} residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in VH). Residues 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in _H (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

"Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV) 4 -Fc, di-scFv, di-scFv, or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments," each with a single antigen-binding site, and a residual "Fc fragment," a name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the _VH - _VL dimer. Overall, these six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only three antigen-specific hypervariable regions) has the ability to recognize and bind to an antigen, although with a lower affinity than the complete binding site.

Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by appending several additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is herein the designation for Fab' in which the cysteine residues of the constant domains carry at least one free sulfhydryl group. F(ab') ₂ antibody fragments were originally produced as paired Fab fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be divided into different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA, and IgA _2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv" antibody fragments comprise the _VH and _VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to small antibody fragments with two antigen-binding sites, comprising a heavy-chain variable domain ( _{VH) connected to a light-chain variable domain (VL )} in the same polypeptide chain ( _VH - _VL ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain ( _VH ) and a light chain variable domain ( _VL ), wherein the _VHVL unit has _polyepitopic specificity; antibodies having two or more _VL domains and _VH domains, wherein each _{VHVL unit} binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, antibody fragments that have been covalently or non-covalently linked. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same target or different targets. "Monospecificity" refers to the ability to bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

The expression "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (alpacas and llamas) and cartilaginous fish (e.g., nurse sharks) and those derived from human and mouse antibodies by recombinant methods (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO 03/035694; Febs Lett (1994) 339:285-290; WO 00/29004; WO 02/051870).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is identical and/or in conjunction with the same epitope, except for possible variants that may occur during the production of the monoclonal antibody, and such variants are generally present in a minute amount. In contrast to the polyclonal antibody preparations generally comprising different antibodies for different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are not mixed by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by obtaining from a substantially homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the method provided herein can be produced by the hybridoma method described first by Kohler et al., Nature 256:495 (1975), or can be produced by recombinant DNA methods (see, for example, U.S. Patent number 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

"Monoclonal antibodies" herein specifically include "chimeric antibodies" (immunoglobulins) in which portions of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys such as baboon, rhesus monkey or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

The " humanized " form of non-human (for example, mouse) antibody is a chimeric antibody containing the minimum sequence derived from non-human immunoglobulin. For most cases, humanized antibodies are these human immunoglobulins (receptor antibodies), wherein the residues from the hypervariable region of the receptor are replaced by residues from the hypervariable region with required specificity, affinity and ability of non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can be included in the residues that do not exist in the receptor antibody or in the donor antibody. Make these modifications to further improve antibody performance. Generally, humanized antibodies will comprise at least 1 and generally all of 2 variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin and all or substantially all of the FR regions are those FRs with human immunoglobulin sequences, with the exception of FR replacements as shown above. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

For purposes herein, a "complete antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the complete antibody has one or more effector functions.

"Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bond. Each heavy chain has a variable domain ( _VH ) at one end followed by a number of constant domains. Each light chain has a variable domain ( _VL ) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains.

A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule (eg, a cytotoxic moiety or radiolabel).

In some embodiments, antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. The main cells used to mediate ADCC, namely NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) page 464. In order to assess the ADCC activity of the target molecule, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, eg, in a animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred.

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target cell in the presence of complement. The complement activation pathway begins with the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a human native sequence FcR. In addition, a preferred FcR is a receptor that binds to IgG antibodies (gamma receptors) and includes FcγRI, FcγRII and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

"Contaminants" refer to substances that are different from the desired polypeptide product. In some embodiments of the present invention, contaminants include charge variants of polypeptides. In some embodiments of the present invention, contaminants include charge variants of antibodies or antibody fragments. In other embodiments of the present invention, contaminants include, but are not limited to: host cell substances, such as CHOP; dissolved protein A; nucleic acids; variants, fragments, aggregates or derivatives of the desired polypeptide; another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc. In some examples, the contaminant can be a host cell protein (HCP), which comes from, for example, but not limited to, bacterial cells such as E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells.

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with the desired binding specificity, which is not the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesive protein portion of an immunoadhesin molecule is generally a continuous amino acid sequence that contains at least a receptor or ligand binding site. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

As used herein, "substantially the same" means that a value or parameter has not been changed by a significant action. For example, the ionic strength of the chromatographic mobile phase at the column outlet is substantially the same as the initial ionic strength of the mobile phase if the ionic strength has not been significantly changed. For example, an ionic strength at the column outlet that is within 10%, 5%, or 1% of the initial ionic strength is substantially the same as the initial ionic strength.

Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It will be understood that the various aspects and variations of the invention described herein include "consisting of" and/or "consisting essentially of" aspects and variations.

II. Chromatographic Methods

A. Determine the optimal ion exchange chromatography separation conditions

The present invention provides methods for predicting optimal ion exchange conditions for performing IEC on polypeptides, thereby minimizing the loss of resolution due to changes in pH and temperature. In some embodiments, ion exchange chromatography is used to detect contaminants in compositions comprising polypeptides. In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof. In some embodiments, the contaminant is a charge variant; for example, a basic charge variant and/or an acidic charge variant of a polypeptide, including a basic charge variant and/or an acidic charge variant of an antibody or antibody fragment.

In some embodiments of the present invention, it is determined that the polypeptide is in a condition of charge balance. The net charge state of the polypeptide is plotted against pH to illustrate this balance. The curve is generated using the amino acid sequence of the polypeptide. The area of the curve where the slope is closest to zero represents charge balance. At equilibrium, the net charge state of the polypeptide resists changes due to pH changes, which is graphically displayed as the flattest area on the curve (Figure 1). The stability of the polypeptide charge state contributes to the robustness of the assay. The condition where the polypeptide is in charge balance can be analyzed by setting the second derivative of the straight line equation of z and pH to be equal to 0. This is the inflection point of the curve where the curve changes from concave to convex or from convex to concave. Although there are multiple inflection points (IP) on this curve (not shown in Figure 1), the inflection point of interest is inside the biological region where the absolute value of the slope no longer decreases. This IP produces a significantly robust method, which is attributed to the stability of the charge state relative to pH.

The charge balance of a peptide is ideal for IEC separations because contaminants with slight net charge differences compared to the target peptide can be detected over a range of pH values. This is attributed to the structure and properties of the amino acids that make up the peptide. Six amino acids are used to calculate the net charge state (z) (Table 1) because they play an important role in defining the pH-dependent characteristics of proteins. The acid dissociation constant, pKa, is defined as (-log ₁₀ Ka) and is used to calculate the charge state of an amino acid based on the constant ratio [A-]/[HA]. The result is not an actual value, but rather the probability P of this charge state.

Table 1. Acid dissociation constants of selected amino acids.

Equation 1

For example, using Equation 1 for histidine at pH 6.5, P = 10 ^(6.5-6) / (10 ^(6.5-6) + 1) ≈ 0.76. This means that each histidine residue in a polypeptide containing 10 histidine residues will have a 76% chance of being deprotonated at pH 6.5, rather than a +0.24 charge (1-0.76). In other words, at pH 6.5, approximately three out of every four histidine residues in the polypeptide will be deprotonated. This can be compared to the calculated value for the polypeptide at pH 7.5 (Figure 2B), where nearly all histidine residues are deprotonated. The frequency of the most prevalent charge state decreases as the pH approaches the pKa of the amino acid side chain.

Applying this equation to key amino acids illustrates why operating at equilibrium provides the best separation. Weighting the probabilities of the six charge-determining amino acids over this pH range yields the most uniform charge state ( FIG. 3 ). The presence of different charge states due to the probability distribution of protonated species will obscure the results and hinder the ability to detect contaminants with slightly different net charge distributions compared to the subject polypeptide. In some embodiments, a 3D plot of net charge distribution versus pH is drawn. Higher separation is achieved when the peaks of charge versus pH versus frequency are sharpest ( FIG. 5 ). Thus, identical pH and temperature conditions for robustness produce the best separation.

In some embodiments of the present invention, the charge distribution frequency is determined by Shannon entropy, which is a measure of the uncertainty of a random variable (Equation 2). Based on the number of residues of the six amino acids present in the polypeptide (lysine, histidine, aspartic acid, glutamic acid, tyrosine, and arginine), the Shannon entropy of the polypeptide at a given pH is plotted as a function of pH at a given temperature (Fig. 4). The lower the Shannon entropy, the more uniform the charge distribution. In some embodiments of the present invention, chromatography is performed at a pH and temperature at which the Shannon entropy is approximately minimum.

Equation 2

in:

n = possible outcomes (n = 2, protonation or deprotonation)

P = probability of outcome or event ( _xi ) (see Equation 1)

b = # trials (number of charged amino acid residues)

In some embodiments of the present invention, the optimal ion exchange chromatography separation conditions for a variety of different polypeptides are determined so that a common chromatography method is used to analyze a variety of polypeptide products; for example, a variety of antibody products. In some embodiments, a common chromatography method determined by the methods described herein is used to analyze a variety of polypeptide products (e.g., a variety of antibody products) for the presence of contaminants (such as charge variants). A significant advantage of the present invention is that the IPs of a variety of polypeptides (including a variety of mAbs) appear at the same pH ( FIG. 7 ), differing only by the number of charges at this inflection point. Therefore, the optimal conditions for all IECs of these polypeptides will be the same; that is, at a pH and temperature at which the polypeptides are in charge equilibrium.

In order to ensure that the change in conditions will not cause deviation from the IP, the term dIP/dT value is used. The dIP/dT of a protein is the change in the net charge of the polypeptide to the pH curve inflection point relative to temperature changes. The inflection point of a given polypeptide will be based on the temperature fluctuations of the net charge to pH curve. However, although the inflection point pH decreases with increasing temperature (see, for example, Figures 8 and 9), the net charge remains constant. Therefore, optimizing chromatography for the inflection point also provides ion exchange methods with robustness against temperature fluctuations.

In some embodiments, the present invention provides a means for determining the type of ion exchange chromatography to use for a given polypeptide in a plurality of polypeptides. For example, if the net charge is positive at the inflection point, a cation exchange chromatography material is used. Non-limiting examples of cation exchange chromatography materials are provided below. In some embodiments, a common cation exchange chromatography method is used to analyze a plurality of polypeptides (e.g., antibodies), wherein the plurality of polypeptides have a net positive charge at a common inflection point. If the net charge is negative at the inflection point, an anion exchange chromatography material is used. Non-limiting examples of anion exchange chromatography materials are provided below. In some embodiments, a common anion exchange chromatography method is used to analyze a plurality of polypeptides (e.g., antibodies), wherein the plurality of polypeptides have a net negative charge at a common inflection point. B. Determining the Optimal Buffer System

In some embodiments, the present invention provides methods for selecting the optimal buffer for use in a chromatography method. In some embodiments, a buffer system is used in the chromatography method with a rate of change of the acid dissociation constant (pKa) that is similar to the rate of change of the inflection point and the temperature change. Selecting a buffer in which the change in pKa relative to the temperature change (i.e., dpKa/dT) is approximately equal to the dIP/dT of the protein ensures that any temperature change will allow the protein to remain at IP, thereby contributing to the robustness of the analytical chromatography. In some embodiments, (dIP/dT) _polypeptide ≈ (dpKa/dT) _buffer . In some embodiments, the buffer is an ACES buffer or a HEPES buffer. For example, using ACES or HEPES buffer, the charge state of the exemplary mAb at the inflection point varies by less than 0.5 above 30°C (Figure 11).

Equation 3dIP/dT≈dpKa/dT

In some embodiments, the buffer provides effective buffering capacity at the inflection point pH. In some embodiments, the dIP/dT of the polypeptide is about -0.02. In some embodiments, the temperature change is from about 20°C to about 50°C. In some embodiments, dIP/dT=dpKa/dT±1%, dIP/dT=dpKa/dT±2%, dIP/dT=dpKa/dT±3%, dIP/dT=dpKa/dT±4%, dIP/dT=dpKa/dT±5%, dIP/dT=dpKa/dT±6%, dIP/dT=dpKa/dT±7 %, dIP/dT=dpKa/dT±8%, dIP/dT=dpKa/dT±9%, dIP/dT=dpKa/dT±10%, dIP/dT=dpK a/dT±20%, dIP/dT=dpKa/dT±30%, dIP/dT=dpKa/dT±40%, or dIP/dT=dpKa/dT±50%. In some embodiments, the net charge of the polypeptide in the selected buffer varies by less than 1 above about 5°C, 10°C, 15°C, 20°C, 25°C, or 30°C.

In some embodiments, the present invention provides methods for developing high-resolution and robust multi-product polypeptide IECs to detect contaminants such as charge variants. Conditions are designed such that the polypeptides (e.g., mAbs) are in charge equilibrium to improve the separation of charged variants from the parent polypeptide. The charge balance of multiple polypeptide products (e.g., mAb products) is determined by plotting the calculated net charge state (z) against pH. Conditions in which the polypeptides are in charge equilibrium are resolved by setting the second derivative of the linear equation of z and pH to 0.

Based on the content of six kinds of amino acids in mAb, the net charge of polypeptide at given pH is determined, wherein the six kinds of amino acids play an important role in limiting the pH dependency characteristics of proteins by their side chains. These six amino acids are asparagine, glutamic acid, histidine, tyrosine, lysine and arginine. The acid dissociation constant (pKa, defined as -log ₁₀ Ka) of these six kinds of amino acids is used to calculate net charge state (z) (Table 1). For example, at a pH value lower than 6.02, average histidine is protonated and carries a positive charge, while at a pH value higher than 6.02, average histidine is deprotonated and does not carry a charge. For each of these six kinds of amino acids, the probability of the most common charge state relative to a given pH is determined and based on the respective number of residues of these six kinds of amino acids present in the antibody, the weighted charge probability of mAb1 at a given pH is determined. Charge distribution frequency can also be determined by Shannon entropy, which is a measure of random variable uncertainty (Equation 3). Based on the respective number of residues of these six kinds of amino acids present in the polypeptide, the Shannon entropy of polypeptide at a given pH can be plotted as a function of pH. The lower the Shannon entropy, the more uniform the charge distribution. From this data, the net charge distribution of the polypeptides was plotted as a function of pH and the inflection point (IP) closest to neutral pH was determined. This is the pH with the most uniform charge state and will result in the sharpest peak in the IEC separation. To develop a multi-product IEC solution, the inflection point is determined for multiple target polypeptides (e.g., target MAbs) with different pIs. Targeting this IP can improve the pH robustness of IEC.

The term dIP/dT represents the change in the molecular IP with temperature. From these results, an optimal buffer can be selected where the change in the acid dissociation constant of the buffer with temperature approaches dIP/dT (i.e., dIP/dT ≈ dpKa/dT) to minimize temperature effects and improve assay robustness. For a number of buffers, published values for the change in pKa with temperature (dpKa/dT) are as follows: phosphate: -0.0028; HEPES: -0.014; ACES: -0.02; Tris: -0.028; Bicine: -0.018; Tris: -0.021; TAPS: -0.02; and CHES: -0.018 (Benyon, RJ and Easterby, JS, Buffer Solutions The Basics, IRL Press, 1996).

In some aspects, the present invention provides a method for analyzing a plurality of antibody compositions, wherein each antibody composition comprises an antibody and one or more charge variants of the antibody, wherein the method effectively separates the antibody from its charge variant; for each antibody composition, the method comprises, a) using a loading buffer with a flow rate of about 1 mL/minute, binding the antibody and one or more charge variants to an ion exchange chromatography material, wherein the loading buffer comprises 10 mM HEPES buffer at about pH 7.6 at about 40° C.; b) eluting the antibody and charge variant contaminants from the ion exchange chromatography material using a gradient of elution buffer, wherein the elution buffer comprises about 10 mM HEPES buffer at about pH 7.6 and NaCl, wherein the concentration of NaCl in the gradient increases from about 0 mM to about 80 mM in about 40 minutes, wherein the antibody and its charge variant are separated by the gradient; and c) detecting the antibody and one or more charge variants. In some embodiments, the plurality of antibody compositions comprise different antibodies. In some embodiments, the plurality of antibody compositions comprise antibodies with different pIs.

C. Chromatography

In some aspects, the invention provides methods for analyzing a composition comprising a polypeptide and one or more contaminants (e.g., polypeptide variants), the method comprising combining the polypeptide and one or more contaminants with an ion exchange chromatography material using a loading buffer having an initial ionic strength; eluting the polypeptide and one or more contaminants from the ion exchange column using an elution buffer, wherein the ionic strength of the elution buffer is varied by an ionic strength gradient such that the polypeptide and one or more contaminants are eluted from the chromatography material as distinct separate entities. In some embodiments, the chromatography method is suitable for a variety of polypeptides (e.g., polypeptide products) having different pIs. For example, the method can be used for a variety of different antibody products having a pI ranging from 6.0 to 9.5. In other embodiments, the chromatography method comprises using an optimal buffer determined by the methods described herein.

In some embodiments of any method as described herein, the chromatography material is a cation exchange material. In some embodiments, when polypeptide is positively charged at the inflection point as described herein, a cation exchange material is used. In some embodiments, the cation exchange material is such a solid phase, and the solid phase is negatively charged and has free cations so as to exchange cations with the aqueous solution through or through the solid phase. In some embodiments of any method as described herein, the cation exchange material can be a membrane, a monolith (monolith) or a resin. In some embodiments, the cation exchange material can be a resin. The cation exchange material can include a carboxylic acid functional group or a sulfonic acid functional group, such as but not limited to sulfonate, carboxyl, carboxymethyl sulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfinylethyl or orthophosphate. In some embodiments above, the cation exchange chromatography material is a cation exchange chromatography column. In some embodiments, the cation exchange chromatography material is used for different polypeptides with a pI ranging from about 7.0 to about 9.5, such as different antibodies or their fragments. In some embodiments, the cation exchange chromatography material is used for the chromatography method using the best buffer determined by the method as described herein.

Examples of cation exchange materials are known in the art and include, but are not limited to, Mustang S, Sartobind S, SO3Monolith, S Ceramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Sepharose XL (SPXL), CM Sepharose Fast Flow, Capto S, Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. In some embodiments of any of the methods described herein, the cation exchange material is Poros HS50. In some embodiments, the Poros HS resin can be Poros HS 50 μm or Poros HS 20 μm particles. Examples of cation exchange chromatography columns for use in the methods of the present invention include, but are not limited to, ProPac WCX-10, ProPac WCX-10HT, MabPac SCX-10 5 μm, and MabPac SCX-10 10 μm.

In some embodiments of any method described herein, the chromatography material is an anion exchange material. In some embodiments, when the polypeptide is negatively charged at the inflection point as described herein, an anion exchange material is used. In some embodiments, the anion exchange chromatography material is such a solid phase, which is positively charged and has free anions so as to exchange anions with an aqueous solution passing through or through the solid phase. In some embodiments of any method described herein, the anion exchange material can be a membrane, a monolith or a resin. In one embodiment, the anion exchange material can be a resin. In some embodiments, the anion exchange material can include primary amine, secondary amine, tertiary amine or quaternary ammonium ion functional groups, polyamine functional groups or diethylaminoethyl functional groups. In some of the above-mentioned embodiments, the anion exchange chromatography material is an anion exchange chromatography column. In some embodiments, the anion exchange chromatography material is for example used for polypeptides with a pI less than about 7, such as antibodies or their fragments. In some embodiments, the anion exchange chromatography material is used for different polypeptides with a pI ranging from about 4.5 to about 7.0, such as different antibodies or their fragments. In some embodiments, anion exchange chromatography materials are used in chromatography methods using an optimal buffer determined by the methods described herein.

Examples of anion exchange materials are known in the art and include, but are not limited to, Poros HQ 50, Poros PI 50, Poros D, Mustang Q, Q Sepharose FF, and DEAE Sepharose. Examples of anion exchange chromatography columns for use in the methods of the present invention include, but are not limited to, Dionex ProPac 10SAX and Tosoh GSKgel Q STAT 7μMWAX.

In some embodiments of any method described herein, the chromatography material is a mixed mode material comprising functional groups capable of performing one or more of the following functions: anion exchange, cation exchange, hydrogen bonding, and hydrophobic interaction. In some embodiments, the mixed mode material comprises functional groups capable of performing anion exchange and hydrophobic interaction. The mixed mode material can contain N-benzyl-N-methylethanolamine, 4-mercapto-ethyl-pyridine, hexylamine, or phenylpropylamine as a ligand or contain cross-linked polyallylamine. Examples of mixed mode materials include Capto Adhere resin, QMA resin, Capto MMC resin, MEP HyperCel resin, HEA HyperCel resin, PPA HyperCel resin, or ChromaSorb membrane or Sartobind STIC. In some embodiments, the mixed mode material is Capto Adhere resin. In some of the above-mentioned embodiments, the mixed mode material is a mixed mode chromatography column.

In some embodiments of any of the methods described herein, the ion exchange material can utilize conventional chromatography materials or counter-current chromatography materials. Conventional chromatography materials include, for example, permeability materials (e.g., poly(styrene-divinylbenzene) resins) and diffusivity materials (e.g., cross-linked agarose resins). In some embodiments, the poly(styrene-divinylbenzene) resin can be a Poros resin. In some embodiments, the cross-linked agarose resin can be a sulfopropyl-sepharose fast flow ("SPSFF") resin. The counter-current chromatography material can be a membrane (e.g., polyethersulfone) or a monolithic material (e.g., a cross-linked polymer). The polyethersulfone membrane can be Mustang. The cross-linked polymer monolithic material can be a cross-linked poly(glycidyl methacrylate-co-ethyl dimethacrylate).

In some embodiments of any of the methods of the invention, the chromatography material is in a chromatography column; for example, in a cation exchange chromatography column or an anion exchange chromatography column. In some embodiments, the chromatography column is used for liquid chromatography. In some embodiments, the chromatography column is used for high performance liquid chromatography (HPLC). In some embodiments, the chromatography column is an HPLC chromatography column; for example, a cation exchange HPLC column or an anion exchange HPLC column.

An exemplary HPLC method that can be used for the multi-product chromatography method of the present invention is as follows; however, the method of the present invention should not be construed as being limited by these methods. The sample is added to an autosampler and refrigerated (5 ± 3°C). The column is placed in a column oven and a temperature control feature can be used to maintain the column oven temperature within a narrow range (± 1°C) from the set point during analysis. The column effluent is monitored at 280nm.

The sample is diluted with mobile phase to a target polypeptide concentration of approximately 1-2 mg/mL. In some embodiments, the polypeptide can be digested with carboxypeptidase B (CpB) added at a 1:100 (w/w) ratio and incubated at 37°C for 20 minutes. The sample can be stored at 5°C until analysis.

The instrument may include a low-pressure quaternary gradient pump, a rapid separation autosampler with temperature control capability, a thermally controlled column oven, and a diode array UV detector. At the detector outlet, a PCM-3000 pH and conductivity monitor may be connected to collect pH and conductivity data in real time. Instrument control, data acquisition, and data analysis may be performed, for example, using Thermo Scientific Dionex Chromeleon version 6.8.

The sample was diluted to 2 mg/mL with deionized water and maintained at 5 ± 3 ° C in an autosampler. A MabPac SCX-10, 4 × 250 mm was placed in a column oven set at 37 ± 1 ° C. For each chromatography run, 10 μL of protein (20 μg) was injected. Buffer A was 5 mM ACES pH 7.5 at 37 ° C. Buffer B was 180 mM NaCl in buffer A. By mixing buffer B into buffer A, the gradient was 0-100 mM NaCl at 1 mM/min over 100 minutes. The flow rate was 0.8 mL/min. Protein was detected by absorbance at 280 nm. In some embodiments, buffer A was 10 mM HEPES buffer pH 7.6 at 40 ° C and buffer B was 100 mM NaCl.

As used herein, elution is to remove product, such as polypeptide, and or pollutant from chromatography material. Elution buffer is the buffer used to elute polypeptide or other target products from chromatography material. In some embodiments, elution buffer is an integral part of the mobile phase of chromatography. In some embodiments, a composition comprising polypeptide and pollutant is applied to the chromatography material as part of the mobile phase. Subsequently, mobile phase is changed to elute from the chromatography material along with polypeptide and pollutant, causing polypeptide to be separated from pollutant. In many cases, elution buffer has physical characteristics different from loading buffer. In some embodiments, the ionic strength of elution buffer increases during the elution process compared to loading buffer. In some embodiments, chromatography is a multi-product chromatography method. In some embodiments, elution buffer comprises the optimal buffer determined by the method described herein.

In some embodiments, the ionic strength gradient is a salt gradient. In some embodiments, the salt gradient is a gradient from about 0mM salt to about 200mM salt. In some embodiments, the salt gradient is any one of: about 0mM to about 100mM, 0mM to about 60mM, 0mM to about 50mM, 0mM to about 40mM, 0mM to about 30mM, 0mM to about 20mM, 0mM to about 10mM, 10mM to about 200mM, 10mM to about 100mM, 10mM to about 50mM, 10mM to about 40mM, 10mM to about 30mM, 10mM to about 20mM, 20mM to about 200mM, 20mM to about 100mM, 20mM to about 50mM, 20mM to about 30mM, 30mM to about 200mM, 30mM to about 100mM and 30mM to about 50mM.

In some embodiments of the present invention, the ionic strength of the mobile phase (e.g., elution buffer) is measured by the conductivity of the mobile phase. Conductivity refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by means of ion transport. Therefore, as the amount of ions present in the aqueous solution increases, the solution will have a higher conductivity. The basic unit of measurement for conductivity is Siemen (or mho), mho (mS/cm), and a conductivity meter, such as various models of Orion conductivity meters, can be used. Since electrolytic conductivity is the ability of ions in a solution to conduct an electric current, the conductivity of a solution can be changed by changing the concentration of the ions therein. For example, the concentration of the buffer and/or the concentration of the salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be changed to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is adjusted to achieve the desired conductivity.

In some embodiments, the mobile phase for chromatography has an initial conductivity exceeding any of the following values: 0.0 mS/cm, 0.5 mS/cm, 1.0 mS/cm, 1.5 mS/cm, 2.0 mS/cm, 2.5 mS/cm, 3.0 mS/cm, 3.5 mS/cm, 4.0 mS/cm, 4.5 mS/cm, 5.0 mS/cm, 5.5 mS/cm, 6.0 mS/cm, 6.5 mS/cm In some embodiments, the conductivity of the mobile phase is increased during chromatography, for example, by an ionic strength gradient. In some embodiments, the conductivity of the mobile phase at the time of elution exceeds any of the following values: 1.0 mS/cm, 1.5 mS/cm, 2.0 mS/cm, 2.5 mS/cm, 3.0 mS/cm, 3.5 mS/cm, 4.0 mS/cm, 4.5 mS/cm, 5.0 mS/cm, 5.5 mS/cm, 6.0 mS/cm, 6.5 mS/cm, 7.0 mS/cm, In some embodiments, the conductivity of the mobile phase is increased by a linear gradient. In some embodiments, the conductivity of the mobile phase is increased by a step gradient comprising one or more steps.

In some embodiments of any of the methods described herein; for example, a multi-product chromatography method or a chromatography method comprising an optimal buffer determined by the methods described herein, a composition comprising a polypeptide and one or more contaminants is loaded onto a chromatography material in an amount greater than any of: about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, or 50 μg. In some embodiments, the composition is loaded onto a chromatography material at a concentration greater than any of: about 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, and 5.0 mg/mL. In some embodiments, the composition is diluted prior to loading onto the chromatography material; for example, a dilution of 1:1, 1:2, 1:5, 1:10, or greater than 1:10. In some embodiments, the composition is diluted in the mobile phase of the chromatography. In some embodiments, the composition is diluted in loading buffer.

In some embodiments of any of the methods described herein, the flow rate exceeds any of about 0.5 mL/min, 0.6 mL/min, 0.7 mL/min, 0.8 mL/min, 0.9 mL/min, 1.0 mL/min, 1.1 mL/min, 1.2 mL/min, 1.3 mL/min, 1.4 mL/min, 1.5 mL/min, 1.75 mL/min, and 2.0 mL/min.

In some embodiments of the methods described herein, the chromatography material is in a column. In some embodiments, the column is an HPLC column. In some embodiments, the column has any of the following dimensions: 4×50 mm, 4×100 mm, 4×150 mm, 4×200 mm, 4×250 mm, or 2×250 mm.

D. Detection of Charge Variants

In some aspects, the present invention provides methods for detecting polypeptide (e.g., antibody) variants in a composition comprising a polypeptide and one or more variants in terms of polypeptide composition. In some embodiments, variants of the polypeptide are analyzed using ion exchange chromatography separation conditions optimized as described above. In some embodiments, variants of the polypeptide are analyzed using ion exchange chromatography in which the buffer has been optimized as described above. In some embodiments, variants of the polypeptide are analyzed using ion exchange chromatography in which the separation conditions and buffer have been optimized as described above. In some embodiments, the ion exchange chromatography separation conditions and/or buffer are optimized for multiple polypeptides; for example, by determining a common dIP/dT value for one or more target polypeptides (e.g., one or more antibodies). The method comprises using a loading buffer having an initial ionic strength to bind the polypeptide and one or more variants to an ion exchange chromatography material; eluting the polypeptide and one or more contaminants from the ion exchange column using an elution buffer, wherein the ionic strength of the elution buffer is varied by an ionic strength gradient such that the polypeptide and one or more contaminants are eluted from the chromatography material as distinct, separate entities. The chromatographic eluate is then analyzed for the presence of the parent polypeptide and variant. The variant of polypeptide can comprise the acid variant of polypeptide and the basic variant of parent polypeptide.The example of acid variant, i.e. its pI is less than the variant of parent polypeptide pI, includes but not limited to the polypeptide that wherein one or more glutamine and/or asparagine residues are deamidated.The example of basic polypeptide variant, i.e. its pI is greater than the variant of parent polypeptide pI, includes but not limited to the polypeptide that wherein aspartic acid residue has been modified into succinimide moiety.In some embodiments, polypeptide has the pI of scope from about 6.0 to about 9.5.In some embodiments, polypeptide is the antibody with the pI of scope from about 6.0 to about 9.5.

E. Determining the Purity of the Polypeptide in the Composition

In some aspects, the present invention provides a method for determining the purity of a polypeptide in a composition comprising a polypeptide. In some embodiments, the purity of the polypeptide in the composition is analyzed using ion exchange chromatography separation conditions optimized as described above. In some embodiments, the purity of the polypeptide in the composition is analyzed using ion exchange chromatography in which the buffer has been optimized as described above. In some embodiments, the purity of the polypeptide in the composition is analyzed using ion exchange chromatography in which the separation conditions and buffer are optimized as described above. In some embodiments, the purity of the polypeptide in the composition is analyzed using ion exchange chromatography in which the separation conditions and buffer are optimized as described above. In some embodiments, the ion exchange chromatography separation conditions and/or buffer are optimized for multiple polypeptides; for example, by determining a common dIP/dT value for one or more target polypeptides (e.g., one or more antibodies). The method comprises using a loading buffer having an initial ionic strength to bind the polypeptide and one or more contaminants to an ion exchange chromatography material; eluting the polypeptide and one or more contaminants from the ion exchange column using an elution buffer, wherein the ionic strength of the elution buffer is changed by an ionic strength gradient so that the polypeptide and one or more contaminants are eluted from the chromatography material as different separated entities. The purity of the polypeptide can be assessed by determining the ratio of the amount of polypeptide eluted from the chromatography material to the total amount of contaminants (e.g., charge variants) eluted from the chromatography material. In some embodiments, the polypeptide has a pI ranging from about 6.0 to about 9.5. In some embodiments, the polypeptide is an antibody having a pI ranging from about 6.0 to about 9.5.

F. Determining the Stability of Polypeptides in Compositions

In some aspects, the present invention provides a method for determining the stability of a polypeptide in a composition comprising a polypeptide. In some embodiments, the stability of the polypeptide in the composition is determined using ion exchange chromatography in which the separation conditions are optimized as described above. In some embodiments, the stability of the polypeptide in the composition is determined using ion exchange chromatography in which the buffer has been optimized as described above. In some embodiments, the stability of the polypeptide in the composition is determined using ion exchange chromatography in which the separation conditions and buffer are optimized as described above. In some embodiments, the ion exchange chromatography separation conditions and/or buffer are optimized for a variety of polypeptides; for example, by determining a common dIP/dT value for one or more target polypeptides (e.g., one or more antibodies). In some embodiments, samples of a composition comprising a polypeptide are analyzed over time. In some embodiments, the composition is incubated at various times before analysis. In some embodiments, the composition is incubated at a temperature exceeding any one of the following before analysis: about 0°C, 20°C, 37°C, or 40°C. In some embodiments, the composition is incubated for one or more of the following periods of time prior to analysis: 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 6 months, 1 year. The composition is then analyzed by binding the polypeptide and one or more contaminants in the composition to an ion exchange chromatography material using a loading buffer having an initial ionic strength; eluting the polypeptide and one or more contaminants from the ion exchange column using an elution buffer in which the ionic strength of the elution buffer is varied by an ionic strength gradient such that the polypeptide and one or more contaminants elute from the chromatography material as distinct, separate entities. Changes in the polypeptide to contaminant ratio indicate the stability of the polypeptide in the composition. For example, if the polypeptide to contaminant ratio does not change over time, the polypeptide can be considered stable, while a rapid accumulation of contaminants in the composition accompanied by a decrease in the amount of polypeptide indicates that the polypeptide in the composition is less stable. In some embodiments, the stability of the polypeptide in the composition is analyzed using ion exchange chromatography in which the separation conditions are optimized as described above. In some embodiments, the stability of the polypeptide in the composition is analyzed using ion exchange chromatography in which the buffer has been optimized as described above. In some embodiments, the stability of the polypeptide in the composition is analyzed using ion exchange chromatography in which the separation conditions and buffer are optimized as described above. In some embodiments, the ion exchange chromatography separation conditions and/or buffer are optimized for a variety of polypeptides; for example, by determining a common dIP/dT value for one or more target polypeptides (e.g., one or more antibodies). In some embodiments, the polypeptide has a pI ranging from about 6.0 to about 9.5. In some embodiments, the polypeptide is an antibody with a pI ranging from about 6.0 to about 9.5. Examples of polypeptides include, but are not limited to, antibodies and antibody fragments.

G. Peptide Purification

In some aspects, the present invention provides a method for purifying a polypeptide (such as an antibody) from a composition comprising a polypeptide and one or more contaminants. The method comprises optimizing chromatographic separation conditions as described above. In some embodiments, the polypeptide is purified using chromatography in which the buffer has been optimized as described above. In some embodiments, the polypeptide is purified using chromatography in which the separation conditions and buffer are optimized as described above. In some embodiments, the chromatographic separation conditions and/or buffer are optimized for a variety of polypeptides; for example, by determining a common dIP/dT value for one or more target polypeptides (such as one or more antibodies). In some embodiments, the chromatography is an ion exchange chromatography; for example, a cation exchange chromatography or an anion exchange chromatography. In some embodiments, the chromatography is a mixed mode chromatography.

In some embodiments, a loading buffer having a pH at the polypeptide inflection point is used at the chromatography temperature to combine the polypeptide and pollutants with an ion exchange chromatography material or a mixed mode chromatography material. The loading buffer has an initial ionic strength. Elution buffer is used to elute the polypeptide from an ion exchange chromatography medium or a mixed mode chromatography medium. In the elution buffer, the ionic strength of the elution buffer is changed by an ionic strength gradient so that the polypeptide and one or more pollutants are eluted from the chromatography material as different separation entities. Fractions are collected during the elution phase of the chromatography and fractions that do not contain or contain minimal pollutants are collected and used for further processing or for pharmaceutical preparation. The example of polypeptide includes, but is not limited to, antibodies and antibody fragments.

III. Peptides

The polypeptide is provided for use in any ion exchange chromatography method in which separation conditions are optimized as described herein. In some embodiments of the present invention, a composition of the polypeptide is analyzed by ion exchange chromatography. Such methods can be used to determine the charge variants of the polypeptide within the composition. In some embodiments, the polypeptide is an antibody or fragment thereof. In some embodiments, the polypeptide has a pI ranging from about 6.0 to about 9.5. In some embodiments, the polypeptide is an antibody with a pI ranging from about 6.0 to about 9.5. In some embodiments, the charge versus pH curve inflection point (IP) of the polypeptide is provided by the method of the present invention. In some embodiments, the change in IP over temperature (dIP/dT) is provided by the method of the present invention.

In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is an immunoadhesin.

In some embodiments, the polypeptide has a molecular weight greater than about any of 5,000 daltons, 10,000 daltons, 15,000 daltons, 25,000 daltons, 50,000 daltons, 75,000 daltons, 100,000 daltons, 125,000 daltons, or 150,000 daltons. The polypeptide can have a molecular weight of any of about 50,000 daltons to 200,000 daltons or 100,000 daltons to 200,000 daltons. Alternatively, the polypeptide used herein can have a molecular weight of about 120,000 daltons or about 25,000 daltons.

pi is the isoelectric point and is the pH at which a particular molecule or surface carries no net charge. In some embodiments, the methods of the present invention can be used for a variety of compositions comprising polypeptides, wherein the pi of the polypeptide (e.g., an antibody) in the composition ranges from about 6.0 to about 9.5. In some embodiments, the polypeptide has a pi greater than about 9.5; for example, from about 9.5 to about 12. In some embodiments of any of the methods described herein, the pi of the polypeptide (e.g., an antibody) can be less than about 7; for example, from about 4 to about 7.

In embodiments of any of the methods described herein, one or more contaminants in a composition comprising a polypeptide and one or more contaminants is a polypeptide charge variant. In some embodiments, a polypeptide charge variant is a polypeptide that has been modified from its native state such that the charge of the polypeptide is altered. In some embodiments, the charge variant is more acidic than the parent polypeptide; that is, it has a lower pI than the parent polypeptide. In other embodiments, the charge variant is more basic than the parent polypeptide; that is, it has a higher pI than the parent polypeptide. In some embodiments, the polypeptide charge variant is engineered. In some embodiments, the polypeptide charge variant is the result of natural processes, such as oxidation, deamidation, processing of C-terminal lysine residues, N-terminal pyroglutamate formation, and glycation. In some embodiments, the polypeptide charge variant is a glycoprotein, wherein the glycans attached to the protein are modified such that the charge of the glycoprotein is altered compared to the parent glycoprotein; for example, due to the addition of sialic acid or a derivative thereof. In some embodiments, the polypeptide charge variant is an antibody charge variant.

The polypeptide to be analyzed using the methods described herein is generally produced using recombinant technology. The method for producing recombinant proteins is described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are incorporated herein by reference. In some embodiments, the target protein is produced in CHO cells (see, for example, WO 94/11026). In some embodiments, the target polypeptide is produced in Escherichia coli cells. See, for example, U.S. Patent No. 5,648,237; U.S. Patent No. 5,789,199, and U.S. Patent No. 5,840,523, which describe translation initiation regions (TIR) and signal sequences for optimizing expression and secretion. Also referring to Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C.Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describe expression of antibody fragments in E. coli. When using recombinant techniques, the polypeptide can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium.

The polypeptide can be recovered from the culture medium or from the host cell lysate. The cells used in expressing the polypeptide can be destroyed by a variety of physical or chemical means, such as freeze-thaw cycles, ultrasonic treatment, mechanical disruption or cell lysates. If the polypeptide is produced intracellularly, as a first step, the granular debris (host cells or cracked fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for separating polypeptides secreted into the periplasmic space of Escherichia coli. In short, the cell paste is melted in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. The cell debris is removed by centrifugation. In the case of polypeptide secretion into the culture medium, a commercially available protein concentration filter, for example, Amicon or Millipore Pellicon ultrafiltration device, is usually used first to concentrate the supernatant from this type of expression system. Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

In some embodiments, the polypeptide in a composition comprising the polypeptide and one or more contaminants has been purified or partially purified prior to analysis by the methods of the present invention. For example, the polypeptide of the methods is an eluent from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography. In some embodiments, the polypeptide is in the eluent from Protein A chromatography.

Examples of polypeptides that can be analyzed by the methods of the present invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, and interleukins. (A) Antibodies

In some embodiments of any of the methods described herein, the polypeptide used in any of the methods of analyzing polypeptides and preparations comprising polypeptides by the methods described herein is an antibody.

Molecular targets of the antibodies include CD proteins and their ligands, such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD34, CD40, CD79α (CD79a), and CD79β (CD79b); (ii) ErbB receptor family members such as EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM -1, VCAM and αv/β3 integrins, including their α or β components (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); (iv) growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; C protein, BR3, c-met, tissue factor, β7, etc.; and (v) cell surface and transmembrane tumor-associated antigens (TAAs), such as those described in U.S. Patent No. 7,521,541.

Other exemplary antibodies include those selected from the group consisting of, and not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2/neu antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, Anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-κ light chain antibody, anti-λ light chain antibody, anti-melanosome s antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody and anti-Tn-antigen antibody.

(i) Monoclonal antibodies

In some embodiments, the antibody is a monoclonal antibody. A monoclonal antibody is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants that arise during the production of the monoclonal antibody, such variants generally being present in minor amounts. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as not being a mixture of individual or polyclonal antibodies.

For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methods (US Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to stimulate lymphocytes that produce or are capable of producing antibodies, wherein the antibodies specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

In some embodiments, myeloma cells are these myeloma cells, and they efficiently merge, support the cell stabilization of the antibody production selected to produce antibodies at a high level and are sensitive to culture medium (such as HAT culture medium). Among these cells, in some embodiments, myeloma cell line is mouse myeloma line, such as those derived from MOPC-21 and MPC-11 mouse tumors obtained from Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockwell, Maryland, USA. Human myeloma and mouse-human heterozygous myeloma cell lines (Kozbor, J. Immunol, 133: 3001 (1984) for producing human monoclonal antibodies have also been described; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51-63 pages (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is analyzed for the production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity are identified, these clones can be subcloned by limiting dilution and grown by standard methods (Goding, Brodeur et al., Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors.

The monoclonal antibodies secreted by these subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The DNA encoding the monoclonal antibody is easily isolated using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). In some embodiments, hybridoma cells serve as a source of such DNA. Once separated, this DNA can be placed into an expression vector, which is then transfected into a host cell that does not produce immunoglobulin polypeptides, such as an Escherichia coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Reviews of recombinantly expressed DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol. 5: 256-262 (1993) and Plückthun, Immunol. Revs., 130: 151-188 (1992).

In another embodiment, antibodies or antibody fragments can be separated from antibody phage libraries produced using the technology described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991) describe the use of phage libraries to separate mouse antibodies and human antibodies. Subsequent publications describe the production of high-affinity (nM level) human antibodies (Marks et al., Bio/Technology 10: 779-783 (1992)) by chain shuffling, and combinatorial infection and in vivo recombination as strategies for building very large phage libraries (Waterhouse et al., Nuc. Acids. Res, 21: 2265-2266 (1993)). Thus, these technologies are effective alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

This DNA also may be modified, for example, by replacing the coding sequences for human heavy and light chain constant domains with the homologous mouse sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)) or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site with specificity for one antigen and another antigen-combining site with specificity for a different antigen.

In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

(ii) Humanized antibodies

In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, humanized antibodies have been introduced into one or more amino acid residues from non-human sources. These non-human amino acid residues are often referred to as "input" residues, which are generally taken from "input" variable domains. Humanization can be carried out by replacing the hypervariable region sequence with the corresponding human antibody sequence according to Winter and collaborators (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Therefore, such humanized antibodies are chimeric antibodies (U.S. Patent number 4,816,567), in which the region substantially smaller than the complete human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The selection of human variable domains (heavy chain and light chain variable domains) to be used for producing humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domains of rodent antibodies is screened for the complete library of known human variable domain sequences. Subsequently, the human sequence closest to the rodent sequence is identified and accepted as the human framework region (FR) for humanized antibodies (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196: 901 (1987)). Another method uses a special framework region derived from the consensus sequence of all human antibodies with a specific subgroup light chain variable region or heavy chain variable region. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

It is also important that the antibody should be humanized, retaining high affinity and other favorable biological properties for the antigen simultaneously. To achieve this goal, in some embodiments of the present method, by using the three-dimensional model of the parental sequence and the humanized sequence, the method for analyzing the parental sequence and the multiple conceptual humanized products is prepared humanized antibody. The three-dimensional immunoglobulin model is normally obtainable and is familiar to those skilled in the art. The computer program that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence is obtainable. The inspection of these display results allows analysis of the possible role that residues play a role in the candidate immunoglobulin sequence, that is, analyzing the residues that affect the ability of the candidate immunoglobulin in conjunction with its antigen. In this way, FR residues can be selected and combined from receptor sequence and input sequence, thereby realizing the antibody characteristics desired, as the affinity increased to the target antigen. Usually, the hypervariable region residues directly and the overwhelming majority participate in affecting the antigen binding effect.

(iii) Human antibodies

In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be produced. For example, transgenic animals (e.g., mice) can now be produced, wherein the transgenic animals, once immunized, are able to produce a complete human antibody library in the absence of endogenous immunoglobulin production. For example, homozygous deletions of antibody heavy chain hinge region (J _H ) genes have been described in chimeric and germline mutant mice, resulting in complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays to such germline mutant mice will result in the production of human antibodies once antigen attack occurs. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immun. 7: 33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and human antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. According to this technology, antibody V domain genes are cloned in frame into the major capsid polypeptide gene or the minor capsid polypeptide gene of a filamentous phage (such as M13 or fd) and displayed on the surface of the phage particle as functional antibody fragments. Because the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that display these properties. Thus, phage mimics some of the characteristics of B cells. Phage display can be performed in a variety of formats; for their review, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V-gene segments from several sources can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse set of anti-oxazolone antibodies from a small random combinatorial library of V genes in the spleen of immune mice. V gene libraries can be constructed from unimmunized human donors, and antibodies against diverse antigen groups (including self-antigens) can be separated substantially according to the techniques described by Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

(iv) Antibody fragments

In some embodiments, the antibody is an antibody fragment. A variety of technologies for producing antibody fragments have been developed. Traditionally, these fragments are derived from intact antibodies by proteolytic digestion (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be directly produced by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another method, F(ab') ₂ fragments can be directly isolated from recombinant host cell cultures. Other technologies for producing antibody fragments will be apparent to technicians. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Antibody fragments may also be "linear antibodies," as described, for example, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab') ₂ fragment, a scFv, a Fv, and a diabody.

(v) Bispecific antibodies

In some embodiments, antibody is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificity to at least two different epitopes. Exemplary bispecific antibodies can be combined with two different epitopes. Alternatively, the bispecific antibody binding arm can be combined with the arm that is bound to the triggering molecule on the leukocyte, such as T cell receptor molecules (such as CD2 or CD3) or IgG Fc receptors (FcγR) such as FCγRI (CD64), FCγRII (CD32) and FcγRIII (CD16), so that the cellular defense mechanism is focused on the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (for example, F (ab') ₂ bispecific antibodies).

Methods for producing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random distribution of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, in which only one molecule has the correct bispecific structure. The purification of the correct molecule is quite cumbersome, usually carried out by affinity chromatography steps, and the product yield is low. Similar methods are disclosed in WO 93/0882 and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. In some embodiments, this fusion has an immunoglobulin heavy chain constant domain, including at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if desired) immunoglobulin light chain is inserted into an independent expression vector and co-transfected into a suitable host organism. In embodiments where the three polypeptide chains used in the construction have different ratios to provide the best yield, this provides flexibility in regulating the mutual ratio of the three polypeptide fragments. However, when at least two polypeptide chains result in high yields with equal expression ratios or when the ratio has no specific meaning, the coding sequences of two or all three polypeptide chains may be inserted into one expression vector.

In some embodiments of this scheme, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure promotes the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations because the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation method. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

According to another scheme described in U.S. Patent number 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises at least a portion of the _CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensation "hole" with the same or similar size as the bulky side chain is produced on the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer productivity relative to other unwanted products (e.g., homodimers).

Bispecific antibodies include cross-linked antibodies or "heterogeneous conjugate" antibodies. For example, one of the antibodies under heterogeneous conjugate can be coupled to avidin, and the other antibody is coupled to biotin. For example, such antibodies have been proposed to guide target immune system cells to unwanted cells (U.S. Patent number 4,676,980) and to treat HIV infection (WO 91/00360, WO92/200373 and EP03089). Any convenient cross-linking method can be used to produce heterogeneous conjugate antibodies. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent number 4,676,980 together with many cross-linking techniques.

The technology of producing bispecific antibodies from antibody fragments has also been described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229:81 (1985) described a method in which intact antibodies are cleaved by proteolysis to produce F(ab') ₂ fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent that stabilizes vicinal dithiols and prevents the formation of intermolecular disulfides. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. Subsequently, one of the Fab'-TNB derivatives is converted back into a Fab'-sulfhydryl group by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab'-TNB derivatives to form a bispecific antibody. The resulting bispecific antibodies can be used as a material for selective enzyme immobilization.

A variety of techniques for directly producing and isolating bispecific antibody fragments from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This approach can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for producing bispecific antibody fragments. These fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker, wherein the linker is too short to allow pairing between the two domains on the same chain. Thus, the _VH and _VL domains of one fragment are forced to pair with the complementary VL and _VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for generating bispecific antibody fragments by utilizing single-chain _Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

Antibodies with greater than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

(v) Multivalent Antibodies

In some embodiments, the antibody is a multivalent antibody. Compared with bivalent antibodies, multivalent antibodies can be internalized (and/or alienated) faster by cells expressing antigens bound to the antibody. The antibodies provided herein can be multivalent antibodies (e.g., tetravalent antibodies) (except the IgM class) with three or more antigen binding sites, which can be easily produced by recombinantly expressing nucleic acids encoding the polypeptide chains of the antibodies. Multivalent antibodies can include a dimerization domain and three or more antigen binding sites. Preferred dimerization domains include or consist of an Fc region or hinge region. In this case, the antibody will include an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. Preferred multivalent antibodies herein include or consist of 3 to about 8, but preferably 4 antigen binding sites. The multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is an Fc region polypeptide chain, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably 4) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise from about 2 to about 8 light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and, optionally, further comprise a CL domain.

In some embodiments, the antibody is a multispecific antibody. Examples of such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain ( _VH ) and _a light chain variable domain ( _VL ), wherein _{the VHVL} unit has polyepitopic specificity; antibodies having two or more _VL domains and _VH domains, wherein each _VHVL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and antibody fragments that have been covalently or non-covalently linked. In some embodiments, the antibody has polyepitopic specificity, e.g., the ability to specifically bind to two or more different epitopes on the same target or different targets. In some embodiments, the antibody is monospecific; e.g., an antibody that binds only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

(vi) Other antibody modifications

It may be desirable to modify the antibodies provided herein with respect to effector functions, such as to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or in addition, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds within the region. The homodimeric antibodies thus produced can have improved internalization capacity and/or improved complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions and thus have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half-life of an antibody, amino acid changes can be made in the antibody as described in US 2006/0067930, which is hereby incorporated by reference in its entirety.

(B) Polypeptide Variants and Modifications

Amino acid sequence modifications of the polypeptides described herein (including antibodies) can be used in methods for purifying the polypeptides described herein (eg, antibodies).

(i) Variant polypeptides

"Polypeptide variant" means a polypeptide as defined herein, preferably an active polypeptide, having at least about 80% amino acid sequence identity with the full-length native sequence of the polypeptide, a polypeptide sequence lacking a signal peptide, or the extracellular domain of the polypeptide with or without a signal peptide. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of such a full-length native amino acid sequence. Typically, TAT polypeptide variants will have at least about 80% amino acid sequence identity, alternatively at least the following percentages of amino acid sequence identity with the full-length native sequence of the polypeptide, a polypeptide sequence lacking a signal peptide, or the extracellular domain of the polypeptide with or without a signal peptide: about 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Optionally, the variant polypeptide will have no more than one conservative amino acid substitution compared to the native polypeptide sequence, alternatively, no more than about any of the following conservative amino acid substitutions compared to the native polypeptide sequence: 2, 3, 4, 5, 6, 7, 8, 9 or 10.

For example, when compared to a full-length native polypeptide, a variant polypeptide may be truncated at the N-terminus or C-terminus, or may lack internal residues. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncation, deletion, and insertion can be prepared by any of a variety of conventional techniques. The desired variant polypeptide can be chemically synthesized. Another suitable technique involves isolating and amplifying nucleic acid fragments encoding the desired variant polypeptides by polymerase chain reaction (PCR). In PCR, oligonucleotides that limit the desired ends of the nucleic acid fragment are used at 5' primers and 3' primers. Preferably, the variant polypeptide shares at least one biological activity and/or immunological activity with the native polypeptide disclosed herein.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include fusions of the N- or C-terminus of the antibody to an enzyme or a polypeptide that increases the serum half-life of the antibody.

For example, it may be desirable to improve the binding affinity and/or other biological properties of the polypeptide. Amino acid sequence variants of the antibody may be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or by peptide synthesis. Such modifications include, for example, deleting residues from the amino acid sequence of the polypeptide and/or inserting residues into the amino acid sequence and/or replacing residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions may be used to obtain the final construct, as long as the final construct has the desired characteristics. Amino acid changes may also alter post-translational processes of the polypeptide (e.g., antibody), such as changing the number or position of glycosylation sites.

Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the polypeptide to the sequences of known homologous polypeptide molecules and minimizing the number of amino acid sequence changes made within regions of high homology.

A useful method for identifying certain residues or regions in a polypeptide (e.g., an antibody) that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or group of target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Amino acid sites that show functional sensitivity to the replacement are then modified by introducing additional or other variants at the replacement site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or target region, and the expressed antibody variants are screened for the desired activity.

Another type of variant is an amino acid substitution variant. These variants replace at least one amino acid residue in the antibody molecule with a different residue. The sites of greatest interest for substitutional mutagenesis include hypervariable regions, but FR variations are also contemplated. Conservative substitutions are shown under the heading "preferred substitutions" in Table 2 below. If such substitutions result in a change in biological activity, then more obvious changes such as those described further below with reference to amino acid classes, such as those in Table 2, may be introduced and the product screened.

Table 2.

Original residue Exemplary substitutions Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn；Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

Substituents that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, e.g., a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain can be used to significantly modify the biological properties of the polypeptide. Amino acids can be grouped according to the similarity of their side chain properties (adapted from A. L. Lehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) Uncharged polarity: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)

(3) Acidic: Asp (D), Glu (E)

(4) Basic: Lys(K), Arg(R), His(H)

Alternatively, naturally occurring residues can be divided into groups based on shared side-chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu; Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain direction: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will exchange a member of one of these classes for a member of another class.

Any cysteine residue that is not involved in maintaining the correct conformation of the antibody can also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the polypeptide to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred substitution variant type involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are produced. A convenient way to produce such substitution variants involves affinity maturation using phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of the M13 packaged inside each particle in a monovalent form from filamentous phage particles. Subsequently, as disclosed herein, the phage-displayed variants are screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the target. Such contact residues and adjacent residues are candidates for substitution as described in detail herein. Once such variants are generated, the panel of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another form of amino acid variants of such polypeptides alters the original glycosylation pattern of the antibody. Polypeptides may comprise non-amino acid moieties. For example, polypeptides may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be a deliberate modification resulting from human intervention. Alterations may include deletion of one or more sugar moieties present in the polypeptide and/or addition of one or more glycosylation sites not present in the polypeptide.

Glycosylation of polypeptides is generally either N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, serve as recognition sequences for enzymatic attachment of the sugar moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the following sugars: N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that the antibody contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such variations can also be achieved by adding or substituting one or more serine or threonine residues into the sequence of the original antibody (for O-linked glycosylation sites).

Removal of carbohydrate moieties present on a polypeptide can be achieved chemically or enzymatically, or by mutational replacement of codons encoding amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endoglycosidases and exoglycosidases.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

(ii) Chimeric polypeptides

The polypeptides described herein can be modified in a manner that forms a chimeric molecule comprising the polypeptide, wherein the polypeptide is fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises a fusion of a polypeptide and a tag polypeptide, wherein the tag polypeptide provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically placed at the amino or carboxyl terminus of the polypeptide. Antibodies against the tag polypeptide can be used to detect the presence of such epitope-tagged polypeptides. In addition, the provision of the epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

In an alternative embodiment, the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or a specific region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with the desired binding specificity, which is not the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesive protein portion of an immunoadhesin molecule is generally a continuous amino acid sequence that contains at least a receptor or ligand binding site. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

Ig fusions preferably include a soluble (transmembrane domain deleted or inactivated) form of a polypeptide that replaces at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, _CH2 , and _CH3 regions, or the hinge, _CH1 , _CH2 , and _CH3 regions, of an IgG1 molecule.

(iii) Polypeptide Conjugates

The polypeptides useful in polypeptide formulations may be conjugated to cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates).

Chemotherapeutic agents that can be used to produce such conjugates can be used. In addition, enzymatically active toxins or fragments thereof that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitory protein, curcin, crotin, sapaonaria officinalis inhibitory protein, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides can be used to produce radioconjugated polypeptides. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. Conjugates of polypeptides and cytotoxic agents are generated using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium salt derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) to generate immunoconjugates. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to polypeptides.

Also contemplated herein are the use of conjugates of polypeptides and one or more small molecule toxins such as calicheamicins, maytansinoids, trichothecenes, and CC1065, and toxinically active derivatives of these toxins.

Maytansine is a mitotic inhibitor that plays a role by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (Maytenus serrata). Subsequently, it was found that some microorganisms also produce maytansines, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs have also been conceived. There are a variety of linking groups known in the art for producing polypeptide-maytansine conjugates, for example, including those disclosed in U.S. Patent number 5,208,020. Linking group includes disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups or esterase-labile groups disclosed in the patents determined above, preferably disulfide groups and thioether groups.

Depending on the type of connection, the linker can be connected to the maytansinoid molecule at multiple positions. For example, an ester connection can be formed by reacting with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position with a hydroxyl group, the C-14 position with a hydroxymethyl modification, the C-15 position with a hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, the connection is formed at the C-3 position of maytansinol or a maytansinol analog.

Another conjugate of interest includes a polypeptide conjugate with one or more calicheamicin molecules. The calicheamicin family of antibiotics can cause double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, for example, U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG, and θ ₁ ^I. Another anti-tumor drug that can be conjugated to an antibody is QFA, which is an antifolate. Catheamicin and QFA have intracellular sites of action and do not easily cross the plasma membrane. Therefore, cells take up these drugs through a polypeptide (e.g., antibody)-mediated internalization process, which greatly enhances their cytotoxic effect.

Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozotocin, vincristine, and 5-fluorouracil, a family of drugs collectively known as the LL-E33288 complex, and esperamicins.

In some embodiments, the polypeptide can be a conjugate between the polypeptide and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

In yet another embodiment, a polypeptide (e.g., an antibody) can be conjugated to a "receptor" (e.g., streptavidin) for tumor pretargeting, wherein the polypeptide-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and subsequent administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic drug (e.g., a radionucleotide).

In some embodiments, the polypeptides can also be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic drug) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme that can act on a prodrug in such a way as to convert it into its more active cytotoxic form.

Useful enzymes include, but are not limited to, alkaline phosphatase, which can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatases, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminases, which can be used to convert the non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as serratia proteases, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drugs; β-lactamases, which can be used to convert drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at their amine nitrogens into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs into active free drugs.

(iv) Others

Another type of polypeptide covalent modification includes linking the polypeptide to one of a variety of non-protein polymers, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The polypeptide can be embedded in, for example, microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., ed. (1990).

IV. Obtaining Polypeptides for Use in Formulations and Methods

The polypeptides used in the analytical methods described herein can be obtained using methods well known in the art, including recombinant methods. The following sections provide guidance on these methods.

(A) Polynucleotide

"Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA.

Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues that are believed to possess mRNA for the polypeptide and express it at detectable levels. Thus, polynucleotides encoding polypeptides can be readily obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or by known synthetic methods (e.g., automated nucleic acid synthesis).

For example, the polynucleotide can encode a complete immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region ( _VH ), but also a heavy chain constant region ( _CH ), which generally contains three constant domains: _CH1 , _CH2 , and _CH3 ; and a "hinge" region. In some cases, the presence of a constant region is desired.

Other polypeptides that can be encoded by a polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fvs, scFvs, Fab' and F(ab') ₂ , and "minibodies". Minibodies are (generally) bivalent antibody fragments from which the _CH1 and C _kappa or _CL domains have been excised. Because minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical/diagnostic applications, but because of their bivalency, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only intact antibody molecules, but also antigen-binding antibody fragments of the types discussed above. Preferably, each framework region present in the encoded polypeptide will contain at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region.

VI. Exemplary Embodiments

1. A method for determining optimal ion exchange chromatography separation conditions for analyzing a plurality of compositions, wherein each composition comprises polypeptides together with one or more contaminants, the method comprising a) plotting net charge versus pH curves at a selected temperature based on the amino acid composition of the polypeptides of the two or more compositions, and b) determining the inflection point of the net charge versus pH curves at or near neutral pH by determining the second derivative of the curves of step a); wherein the optimal ion exchange chromatography separation conditions are at a pH at approximately a common inflection point for the polypeptides of the one or more compositions.

2. The method of embodiment 1, wherein if the net charge at the inflection point is positive, a cation exchange material is used for ion exchange chromatography.

3. The method of embodiment 2, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material.

4. The method of embodiment 1, wherein if the net charge at the inflection point is negative, an anion exchange material is used for chromatography.

5. The method of embodiment 4, wherein the anion exchange chromatography material is a quaternary amine chromatography material or a tertiary amine chromatography material.

6. The method of embodiment 1, wherein a mixed-mode chromatography material is used for chromatography.

7. The method of embodiment 6, wherein the mixed-mode ion exchange material is a mixture of a sulfonated chromatography material or a carboxylated chromatography material and a quaternary amine chromatography material or a tertiary amine chromatography material packed in sequence.

8. The method of any one of embodiments 1-7, further comprising c) determining the change in pH of the inflection point of the net charge versus pH curve as a function of temperature (dlP/dT) for the polypeptides of two or more compositions, and d) selecting a buffer for chromatography, wherein the change in the acid dissociation constant of the buffer as a function of temperature (dpKa/dT) is substantially the same as the dlP/dT of the polypeptides.

9. The method of embodiment 8, wherein the buffer provides an effective buffering capacity at the inflection point pH.

10. The method of any one of embodiments 1 to 9, wherein the dIP/dT of one or more polypeptides of the composition is about -0.02 pH units.

11. The method of any one of embodiments 1 to 10, wherein the temperature change is from about 20°C to about 70°C.

12. The method of any one of embodiments 1 to 11, wherein the temperature change is from about 20°C to about 50°C.

13. The method according to any one of embodiments 8 to 12, wherein dpKa/dT = dIP/dT ± 50%.

14. The method according to any one of embodiments 8 to 13, wherein the net charge of the polypeptide in the buffer selected in step d) varies by less than 0.5 above 30°C.

15. The method according to any one of embodiments 8 to 14, wherein the buffer selected in step d) is used in the chromatography at a concentration ranging from about 5 mM to about 250 mM.

16. The method according to any one of embodiments 1 to 15, wherein the buffer composition further comprises a salt.

17. The method of embodiment 16, wherein the salt is NaCl, KCl, (NH ₄ ) ₂ SO ₄ or Na ₂ SO ₄ .

18. The method of embodiment 16 or 17, wherein the concentration of the salt is from about 1 mM to about 1 M.

19. The method according to any one of embodiments 1 to 18, wherein the polypeptide is an antibody or immunoadhesin or a fragment thereof.

20. The method according to any one of embodiments 1 to 19, wherein the polypeptide is a monoclonal antibody or a fragment thereof.

21. The method of embodiment 19 or 20, wherein the antibody is a human antibody.

22. The method of embodiment 19 or 20, wherein the antibody is a humanized antibody.

23. The method of embodiment 19 or 20, wherein the antibody is a chimeric antibody.

24. The method according to any one of embodiments 19 to 23, wherein the antibody is an antibody fragment.

25. The method according to any one of embodiments 1 to 14, wherein the contaminant is a variant of the polypeptide.

26. The method according to any one of embodiments 1 to 25, wherein the contaminant is a degradation product of the polypeptide.

27. The method according to any one of embodiments 1 to 26, wherein the contaminant is a charge variant of the polypeptide.

28. A method for determining optimal ion exchange chromatography separation conditions for analyzing a composition comprising a polypeptide together with one or more contaminants, the method comprising a) plotting a net charge versus pH curve at a selected temperature based on the amino acid composition of the polypeptide, and b) determining the inflection point of the net charge versus pH curve at or near neutral pH by determining the second derivative of the curve of step a); wherein the optimal ion exchange chromatography separation conditions are the pH at about the inflection point of the polypeptide.

29. The method of embodiment 28, wherein if the net charge at the inflection point is positive, a cation exchange material is used for ion exchange chromatography.

30. The method according to embodiment 29, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material.

31. The method of embodiment 28, wherein if the net charge at the inflection point is negative, an anion exchange material is used for chromatography.

32. The method according to embodiment 31, wherein the anion exchange chromatography material is a quaternary amine chromatography material or a tertiary amine chromatography material.

33. The method according to embodiment 28, wherein a mixed-mode chromatography material is used for chromatography.

34. The method of embodiment 33, wherein the mixed-mode ion exchange material is a mixture of a sulfonated chromatography material or a carboxylated chromatography material and a quaternary amine chromatography material or a tertiary amine chromatography material packed in sequence.

35. The method of any one of embodiments 28-34, further comprising c) determining, for the polypeptide, the change in pH of the inflection point of the net charge versus pH curve as a function of temperature (dlP/dT), and d) selecting a buffer for chromatography, wherein the change in the acid dissociation constant of the buffer as a function of temperature (dpKa/dT) is substantially the same as the dlP/dT of the polypeptide.

36. The method of embodiment 35, wherein the buffer provides an effective buffering capacity at the inflection point pH.

37. The method of any one of embodiments 28-36, wherein the dIP/dT of one or more polypeptides of the composition is about -0.02 pH units.

38. The method according to any one of embodiments 28 to 37, wherein the temperature change is from about 20°C to about 70°C.

39. The method of any one of embodiments 28 to 38, wherein the temperature change is from about 20°C to about 50°C.

40. The method of any one of claims 28-39, wherein dIP/dT = dpKa/dT ± 50%.

41. The method according to any one of embodiments 28 to 40, wherein the net charge of the polypeptide in the buffer selected in step d) varies by less than 0.5 above 30°C.

42. The method according to any one of embodiments 28 to 41, wherein the buffer selected in step d) is used in the chromatography at a concentration ranging from about 5 mM to about 50 mM.

43. The method according to any one of embodiments 28 to 42, wherein the buffer composition further comprises a salt.

44. The method of embodiment 43, wherein the salt is NaCl, KCl, (NH ₄ ) ₂ SO ₄ or Na ₂ SO ₄ .

45. The method of embodiment 43 or 44, wherein the concentration of the salt is from about 10 mM to about 1 M.

46. The method according to any one of embodiments 28 to 45, wherein the polypeptide is an antibody or immunoadhesin or a fragment thereof.

47. The method according to any one of embodiments 28 to 46, wherein the polypeptide is a monoclonal antibody or a fragment thereof.

48. The method of embodiment 46 or 47, wherein the antibody is a human antibody.

49. The method of embodiment 46 or 47, wherein the antibody is a humanized antibody.

50. The method of embodiment 46 or 47, wherein the antibody is a chimeric antibody.

51. The method according to any one of embodiments 38-50, wherein the antibody is an antibody fragment.

52. The method according to any one of embodiments 28 to 51, wherein the contaminant is a variant of the polypeptide.

53. The method according to any one of embodiments 28 to 52, wherein the contaminant is a degradation product of the polypeptide.

54. The method according to any one of embodiments 28 to 53, wherein the contaminant is a charge variant of the polypeptide.

55. A method for analyzing a composition, wherein the composition comprises a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) determining optimal pH and temperature ion exchange separation conditions for a plurality of compositions according to the method of embodiment 1, each composition comprising a target polypeptide and one or more contaminants, b) binding the polypeptide and one or more contaminants from the composition to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffer determined by the method of any one of embodiments 8-15; c) eluting the polypeptide and one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises a buffer and a salt, wherein the concentration of the salt increases in the gradient over time, wherein the polypeptide and the one or more contaminants are separated by the gradient; and d) detecting the polypeptide and the one or more contaminants.

56. A method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) binding the polypeptide and one or more contaminants to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffering agent, and wherein the pH and temperature of the chromatography have been optimized for a plurality of target polypeptides by: i) plotting a net charge versus pH curve at a selected temperature, wherein the curve is based on the amino acid composition of the polypeptides of two or more target polypeptides, and ii) determining the inflection point of the net charge versus pH curve by determining the second derivative of the curve of step i); wherein the optimal ion exchange chromatography conditions are the pH at the common inflection point for the two or more target polypeptides; b) eluting the polypeptide and one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises a buffering agent and a salt, wherein the polypeptide and the one or more contaminants are separated by the gradient; and c) detecting the polypeptide and the one or more contaminants.

57. The method of embodiment 56, wherein the selected temperature is ambient temperature.

58. The method of embodiment 56 or 57, wherein the buffer is determined by: a) determining the change in pH as a function of temperature (dIP/dT) of the inflection point of a net charge versus pH curve for two or more target polypeptides, and b) selecting a buffer having a change in acid dissociation constant as a function of temperature (dpKa/dT) that is substantially the same as the dIP/dT of one or more target polypeptides having a common inflection point.

59. The method of embodiment 58, wherein the buffer provides an effective buffering capacity at the inflection point pH.

60. A method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising a) binding the polypeptide and one or more contaminants to an ion exchange chromatography material using a loading buffer, wherein the loading buffer comprises a buffering agent, and wherein the pH and temperature of the chromatography have been optimized for a plurality of target polypeptides; b) eluting the polypeptide and one or more contaminants from the ion exchange chromatography material using a gradient of an elution buffer, wherein the elution buffer comprises a buffering agent and a salt, wherein the polypeptide and one or more contaminants are separated by the gradient; and c) detecting the polypeptide and one or more contaminants.

61. The method of embodiment 60, wherein the buffer is N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES) or (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

62. The method of any one of embodiments 55-61, wherein the concentration of the buffer is from about 5 mM to about 20 mM.

63. The method of any one of embodiments 55-62, wherein the pH of the buffer is from about 6.5 to about 8.5 at a temperature range of about 20°C to about 70°C.

64. The method of any one of embodiments 55-63, wherein the pH of the buffer is from about 6.5 to about 8.5 at a temperature range of about 20°C to about 50°C.

65. The method of any one of embodiments 55-64, wherein the pH of the buffer and polypeptide at the inflection point is about 7.8 at about 22°C, about 7.5 at about 37°C, or about 7.2 at about 50°C.

66. The method according to any one of embodiments 55 to 65, wherein the salt gradient is a linear gradient.

67. The method according to any one of embodiments 55 to 66, wherein the salt gradient is a step gradient.

68. The method according to any one of embodiments 55 to 67, wherein the salt gradient is a NaCl gradient, a KCl gradient, a (NH ₄ ) ₂ SO ₄ gradient or a Na ₂ SO ₄ gradient.

69. The method according to any one of embodiments 55 to 68, wherein the salt concentration in the gradient increases from about 0 mM to about 1 M.

70. The method of embodiment 69, wherein the salt concentration is increased from about 0 mM to about 100 mM in about 100 minutes.

71. The method of embodiment 69, wherein the salt concentration is increased from about 0 mM to about 80 mM in about 40 minutes.

72. The method according to any one of embodiments 55 to 71, wherein the polypeptide is an antibody or immunoadhesin or a fragment thereof.

73. The method according to any one of embodiments 55 to 72, wherein the polypeptide is a monoclonal antibody or a fragment thereof.

74. The method of embodiment 72 or 73, wherein the antibody is a human antibody.

75. The method of embodiment 72 or 73, wherein the antibody is a humanized antibody.

76. The method of embodiment 72 or 73, wherein the antibody is a chimeric antibody.

77. The method of any one of embodiments 72-76, wherein the antibody is an antibody fragment.

78. The method according to any one of embodiments 55 to 77, wherein the contaminant is a variant of the polypeptide.

79. The method according to any one of embodiments 55 to 78, wherein the contaminant is a degradation product of the polypeptide.

80. The method of any one of embodiments 55-79, wherein the contaminant is a charge variant of the polypeptide.

81. The method according to any one of embodiments 55 to 80, wherein the chromatography material is a cation exchange chromatography material.

82. The method according to embodiment 81, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material.

83. A method for analyzing a plurality of polypeptide compositions, wherein each polypeptide composition comprises a polypeptide and one or more charge variants of the polypeptide, wherein the method effectively separates the polypeptide from its charge variants;

For each polypeptide composition, the method comprises a) binding the polypeptide and one or more charge variants to an ion exchange chromatography material using a loading buffer at a flow rate of about 1 mL/minute, wherein the loading buffer comprises 10 mM HEPES buffer at about pH 7.6 at about 40° C.; b) eluting the polypeptide and charge variant contaminants from the ion exchange chromatography material using a gradient of elution buffer, wherein the elution buffer comprises 10 mM HEPES buffer at about pH 7.6 and NaCl, wherein the concentration of NaCl in the gradient increases from about 0 mM to about 80 mM over about 40 minutes, wherein the polypeptide and its charge variants are separated by the gradient; and c) detecting the polypeptide and the one or more charge variants.

84. The method of embodiment 83, wherein the plurality of polypeptide compositions comprises different polypeptides.

85. The method of embodiment 83 or 84, wherein the plurality of polypeptide compositions comprises polypeptides having different pis.

86. The method of any one of embodiments 83-85, wherein the polypeptide composition is an antibody composition.

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

Other details of the present invention are further illustrated by the following non-limiting examples.The disclosures of all references in this specification are expressly incorporated herein by reference.

Example

The following examples are intended to be purely exemplary of the present invention and therefore should not be construed as limiting the invention in any way.The examples and detailed description that follow are offered by way of illustration and not by way of limitation.

Materials and Methods Used in the Examples

Unless otherwise indicated, the following materials and methods were used in the examples.

Material

All mAbs were produced using stable Chinese hamster ovary (CHO) cell lines or Escherichia coli cells.

MabPac SCS-10 and Propac WCX-10 columns were purchased from Thermo Fisher Scientific. AntiBodix columns were from Sepax. YMC columns were purchased from YMC. Trisma (Tris) was obtained from Mallinckrodt Baker Inc. or Sigma (St. Louis, MO), and HEPES, ACES, Trizma base, and CAPS were obtained from Sigma. Sodium chloride, sodium hydroxide (10 N), and hydrochloric acid (12 N) were obtained from Mallinckrodt Baker Inc. Phosphoric acid (85%) was obtained from EMD Millipore.

HPLC setup

Cation exchange chromatography experiments were primarily performed on a Waters 2796 BioAlliance liquid chromatography instrument, an Agilient 1200SL HPLC system, or an UltiMate 3000 Quaternary Rapid Separation LC (ThermoScientific Dionex). The instrumentation included a low-pressure quaternary gradient pump or binary pump, an autosampler with temperature control, a heated column oven for precise temperature control, and a dual-wavelength diode array UV detector. Instrument control, data acquisition, and analysis were performed using Dionex Chromeleon software version 6.8.

Example 1. Optimization of multi-product analytical ion exchange chromatography

To develop high-resolution and robust multi-product IECs for detecting contaminants such as charge variants, conditions were designed such that the mAbs were in charge equilibrium. The charge balance of multiple mAb products was determined by plotting net charge state (z) versus pH. Conditions under which the mAbs were in charge equilibrium were resolved by setting the second derivative of the linear equation of z versus pH to 0.

Based on the content of six kinds of amino acids in mAb, the net charge of mAb at a given pH is determined, wherein the six kinds of amino acids play an important role in limiting the pH-dependent characteristics of proteins by their side chains. These six amino acids are asparagine, glutamic acid, histidine, tyrosine, lysine and arginine. The acid dissociation constant (pKa, defined as -log ₁₀ Ka) of these six kinds of amino acids is used to calculate the net charge state (z). For example, MAb1 has 10 histidine residues. Figure 2 shows the histidine protonation that changes with pH. At a pH value lower than histidine pKa 6.02, histidine is protonated and carries a positive charge (Fig. 2A). At a pH value higher than 6.02, histidine is deprotonated and does not carry a charge. Using equation 1, the probability of a histidine specific charge state is determined when a specific pH is determined. Figure 2 B is presented at pH 6.5 and the possible distribution of deprotonated histidine in mAb1 when pH 7.5. At pH 6.5, mAb1 has a median of 8 deprotonated histidine residues, while at pH 7.5, almost all histidine residues are deprotonated. Figure 2C shows examples of four polypeptide molecules each with two histidine residues. At a pKa (pH 6.02), 50% of the His residues are protonated and 50% are deprotonated. The charge state combinations of the histidine residues on these four molecules exhibit a binomial distribution at the pKa: one molecule has both histidines protonated; two molecules have one histidine protonated and the other deprotonated; and one molecule has both histidines deprotonated.

For each of the six amino acids, the probability of the most common charge state relative to a given pH was determined and the weighted charge probability of mAb1 at a given pH was determined based on the residue number of each of the six amino acids present in the antibody ( FIG. 3 ). The charge distribution frequency can also be determined by the Shannon entropy, which is a measure of the uncertainty of a random variable ( Equation 3 ). The Shannon entropy of mAb1 at a given pH based on the residue number of each of the six amino acids present in mAb1 ( Table 3 ) can be plotted in FIG. The lower the Shannon entropy, the more uniform the charge distribution.

Table 3. Number of selected amino acid residues in mAb1

pH Lysine Histidine Aspartic acid glutamate Tyrosine Arginine Number of residues 90 28 52 64 66 32

From this data, the distribution of the net charge of mAb1 was plotted as a function of pH ( FIG. 5 ) and the inflection point was determined to be pH 7.5 at 37° C. ( FIG. 6 , which is a top view of FIG. 5 ). Note that this is the pH with the most uniform charge state, which appears as the tallest and sharpest peak in FIG. 5 . The most uniform charge state will also result in the sharpest peak in the IEC separation.

Using the above method, the inflection points of various mAbs with pIs ranging from 7.6 to 9.4 were determined ( FIG. 7 ). Surprisingly, the inflection point for almost all mAbs was the same, i.e., pH 7.5 at 37° C. Targeting this IP can improve the pH robustness of IECs. As shown in FIG. 7 , for all antibodies, the net charge varied little between pH 7 and pH 8.

The inflection points of the mAbs were determined at 22° C., 37° C., and 50° C. As shown in FIG8 , although the inflection points were temperature dependent, the inflection points of all antibodies tested were similar at a given temperature.

The inflection point of mAb2 was determined for temperatures ranging from 22°C to 50°C. As shown in Figure 9, although the inflection point pH decreases with increasing temperature, the net charge remains constant. Therefore, optimizing the chromatography for the inflection point also provides the IEX method with robustness against temperature fluctuations. The term dIP/dT represents the change in molecular IP with temperature. From these results, an optimal buffer can be selected where the change in the acid dissociation constant of the buffer with temperature approaches dIP/dT (i.e., dIP/dT ≈ dpKa/dT) to minimize temperature effects and improve assay robustness.

The relationship between the inflection point pH and temperature was plotted and the slope of the linear regression, dpKa/dT values, were calculated for a variety of mAbs with different pI values (Figure 10 and Table 4). The dpKa/dT values for these six mAbs were found to be essentially identical (-0.0177 to -0.0183). Therefore, a buffer with a dpKa/dT value of approximately -0.018 would be optimal for IEC analysis of all mAbs provided in Figure 10 and therefore optimal for multi-product IEC.

Table 4. Inflection Point (pH)

Temperature MAb27 MAb1 MAb2 MAb4 MAb5 MAb6 MAb8 25 7.69 7.73 7.72 7.72 7.75 7.72 7.72 30 7.60 7.64 7.63 7.62 7.66 7.62 7.62 35 7.50 7.54 7.54 7.53 7.56 7.53 7.53 37 7.47 7.51 7.50 7.49 7.53 7.49 7.49 40 7.42 7.45 7.45 7.44 7.47 7.44 7.44 45 7.33 7.37 7.36 7.35 7.39 7.35 7.35 50 7.25 7.28 7.27 7.26 7.30 7.27 7.27 dIP/dT -0.0177 -0.018 -0.018 -0.0183 -0.018 -0.018 -0.018

For a number of buffers, published values for the change in pKa with temperature (dpKa/dT) are as follows:

Phosphate: -0.0028

HEPES: -0.014

ACES: -0.02

Tris: -0.028

N.N-Bicine: -0.018

Tris(hydroxymethyl)methylglycine: -0.021

TAPS: -0.02

CHES: -0.018

See Benyon, RJ and Easterby, JS, Buffer Solutions The Basics, IRL Press, 1996.

Figure 11 shows the temperature-dependent net charge curves at the inflection point for mAb2 in phosphate buffer, HEPES buffer, ACES buffer, and TRIS buffer. The net charge curves for mAb2 in ACES or HEPES buffer are nearly flat, varying by less than 0.5 over a range above 30°C. On the other hand, the curves for Tris and phosphate buffers are less flat, showing greater temperature-dependent changes in net charge. It is concluded that ACES or HEPES buffer is the optimal buffer for multi-product IEC analysis.

Example 2. Formation of a multi-product IEC solution

Based on the relationship between the inflection point and the dpKa/dT values of ACES and HEPES, and the dIP/dT values measured for various mAbs, a multi-product IEC protocol was developed. Nineteen mAbs were tested. mAb samples were diluted to 1 mg/mL in buffer A and maintained in an autosampler at 5 ± 3°C. A MabPac SCX-10, 4 × 250 mm column was placed in a column oven set at 37 ± 1°C. For each chromatography run, 10 μL of protein (20 μg) was injected. Buffer A was 5 mM ACES, pH 7.5, at 37°C. Buffer B was 180 mM NaCl in buffer A. A gradient of 0–100 mM NaCl at 1 mM/min over 100 minutes was applied by mixing buffer B into buffer A. The flow rate was 0.8 mL/min. Proteins were detected by absorbance at 280 nm. As shown in Figure 12, multi-product IEC provided good resolution for a range of diverse mAb products.

Example 3. pH robustness of multi-product IEC

This multi-product IEC was examined for pH robustness using the method described in Example 2, except that the gradient was 1.5 mM NaCl/min and was performed at three different pH values: pH 7.3, pH 7.5, and pH 7.7. mAb4 was used as a non-limiting exemplary antibody for this study.

As shown in Figure 13, good resolution between the main peak of the antibody and its charge variants was seen at all pH values tested. Peak area quantification revealed no significant changes in the analytical results with respect to pH (Table 5).

Table 5. pH robustness of multi-product IEC

^aThe resolution is defined by Equation 4.

Equation 4

Where R is the resolution

t _r1 and t _r2 are the retention times of two adjacent peaks

_w1 and _w2 are the peak widths of two adjacent peaks

Example 4. Temperature Robustness

The temperature robustness of this multi-product IEC was examined using the method described in Example 2, except that the gradient was 1.5 mM NaCl/min and was performed at three different temperatures: 32° C., 37° C., and 42° C. mAb2, mAb6, and mAb10 were used as non-limiting exemplary antibodies for this study.

As shown in Figure 14, for each antibody, good resolution between the main peak of the antibody and its charge variants was seen at all temperatures tested. For each antibody, nearly identical chromatograms were seen at each temperature.

In a second experiment, the temperature robustness of mAbs 19, 7, and 8 was tested in 10 mM HEPES buffer. As shown in Figure 15, for each antibody, good separation between the main peak of the antibody and its charge variants was seen at all temperatures tested. Peak area quantification revealed no significant changes in the analytical results with respect to temperature.

Table 6. Temperature robustness of multiple product IECs

Example 5. Comparison of Multi-Product IEC and Product-Specific IEC

Multi-product IEC was compared to a product-specific IEC method developed for mAb8, mAb25, and mAb26. IEC for mAb8, mAb28, and mAb26 was performed using the multi-product IEC method described in Example 2, except that the gradient was 1.5 mM NaCl/min [Genentech - please confirm]. The buffer and temperature for the product-specific methods were different. For mAb8, it was 20 mM MES pH 6.5 at 30°C; for mAb25, it was 20 mM HEPES pH 7.6 at 42°C; and for mAb26, it was 20 mM ACES pH 7.1 at 40°C. As can be seen in Figure 16, the multi-product IEC (left panel) performed similarly to the product-specific IE method (right panel) or had better resolution.

Example 6. Use of multi-product IEC using different columns

Multi-product IEC was used for chromatography column selection. mAb8 was tested on four different cation exchange columns using the method described in Example 2, except that the gradient was 1.5 mM NaCl/minute. The columns tested were ProPac WCX-10, 4×250, 10 μm; YMC, 4.6×100, 5 μm; Antibodix NP5, 4.6×250, 5 μm; and MabPac SCX-10, 4×250, 10 μm (used in Example 2). As can be seen in Figure 17, all four columns produced adequate resolution. Peak area quantification and the resolution between the acidic and basic peaks and the main peak are shown in Table 7.

Table 7. Columns for screening mAb 8

Example 7. Scalability

The use of cation exchange chromatography columns of different sizes in multi-product IEC was evaluated. Reduced column length will result in shorter run times. Using the multi-product IEC method described in Example 2, mAb8 was chromatographed on three different sizes of ProPacWCX-10 columns. Since the columns had different sizes, the chromatography experiments lasted for different time periods. The column sizes and corresponding run times were as follows: 4×250 mm, 63 minutes, 4×100 mm, 19 minutes, and 4×50 mm, 15 minutes. The results are given in Figure 18. Although some resolution is lost with shorter columns, for high-throughput applications, sufficient separation with consistent quantitative results was obtained with shorter columns. The quantification of peak areas was consistent and is shown in Table 8.

Table 8. Scalability of Multi-Product IEC

Example 8. Robustness of the assay

Validation of testing procedures requires that the method possess appropriate robustness. A design of experiments (DoE) approach to assess robustness comprehensively evaluates the effects of small variations in analytical conditions, including the effects of interactions. Specific multivariate conditions for each experiment are selected to combine factors with the potential for interaction. Factors that cannot be continuously varied but are known to have an effect, i.e., column (e.g., batch-to-batch variability, column age) and instrument (e.g., bimodal type), are tested using a single, single-factor approach. These effects are determined by comparing the variability of the response under the target conditions with the variability of the response under conditions varied according to the factorial design.

Experimental design

Described below is the ion exchange condition for monitoring the recombinant monoclonal antibody protein charge heterogeneity for platform method control scheme.The target of this research is to use six-factor Plackett-Burman experimental design (table 9 and table 10) research determination method robustness.The factor of inspection is solvent pH, salt concentration, column temperature, flow velocity, sample size and buffer mass molar concentration at the end.The response variable of this research includes the relative percentage of main peak, acidic variant and basic variant.A total of 21 tests are carried out, 12 tests are carried out in factor conditions and 9 tests are carried out in target conditions.

Table 13

Table 10

Table 11. Robustness summary table

Statistical analysis

Samples with target response values show the variability that occurs when all variable factors are under the target condition. Samples with factor response values show the variability that occurs when multiple factors are varied in a combined manner.

Table 12

The mean, standard deviation (SD), and relative standard deviation (RSD) were calculated for all target responses and DoE factor responses. Although minor differences were observed between the SD and RSD of target and factor condition isotypes, these were very low. The results are shown in Figures 19-21.

Typically for IEC validation, the acceptable %RSD limits for the main peak are <5% and for the acidic and basic variants are <10%.

All factor conditions produced results for % Main Peak and % Basic Variants that were within the 95/99 tolerance interval calculated from the target condition results.

Two factor conditions #10 (-+--+-) and #12 (-+-+++) produced % acid variant values below the lower limit of the 95/99 TI requirement calculated from the target condition results. All other factor conditions produced values within this confidence interval.

Conditions that yielded values outside the target condition of 95/99 TI were a result of the high level of precision in the assay and limited uncontrolled variability (instrument and column) within the DoE study.

A normal 95/99 TI of IEC may be in the range of 3%-5% for the main peak, acidic variants and basic variants.

Materials and methods

Use the Platform Method Control (PMC) protocol to quantify charged variants of protein or antibody drug substances, drug products, or toxicology materials for clinical products. The Platform Method Control protocol utilizes a representative antibody as a method control to determine system suitability for this multi-product cation exchange chromatography method. This multi-product testing procedure is suitable for protein molecules with a positive net charge (at an approximate pI > 7.2).

Equipment and materials

1.1 HPLC system: Waters UPLC H-Class Bio with tunable UV (TUV); Waters Alliance 2695 with Waters 2487 detector and Waters Alliance e2695 with Waters 2489 UV/Vis detector or equivalent.

1.2 Built-in UV detector capable of monitoring at 280nm.

1.3 The HPLC must contain a column oven capable of maintaining the temperature at ±2°C of the set point.

1,4 Electronic integrator or computer system capable of peak area integration.

1,5 Autosampler capable of cooling to 2-8°C.

1.6 Column: ThermoFisher MabPacR SCX-10, 10 μm, 4250 mm (Thermo, product number 074625).

1.7 pH meter with temperature compensation.

1.8 A water bath capable of heating at 37±2°C.

1.9 A calibrated thermometer with 1°C graduations and intended for use partially immersed in a water bath.

Reagents

Note: Recipes are for nominal amounts of reagents and may be proportionally adjusted according to assay requirements.

2.1 Purified water, suitable for HPLC analysis (Super-Q or equivalent)

2.2 Solvent A: 5 mM HEPES buffer, pH 7.5 ± 0.1

Acid-free HEPES, reagent grade (FW 238.3, Corning CellGro; product number 61-034-RO), 1.87 g

HEPES sodium salt, reagent grade (FW 260.3, Sigma Aldrich; product number H3784), 1.87 g

Purified water to make up to 3L

Combine the listed chemicals with approximately 2900 mL of purified water in a graduated cylinder. Stir until dissolved. Make up to 3 L with purified water and measure the pH. Verify the pH is 7.5 ± 0.1 at ambient temperature. If the pH falls outside the specified range, discard and repeat the preparation. Filter through a 0.2 μm membrane.

2.3 Solvent B: 100 mM NaCl in solvent A

Sodium chloride (FW 58.44 J.T. Baker Cat. No. 3624-01 or equivalent), 5.844 g

Solvent A (step 2.2) is added to 1 L

Combine sodium chloride with approximately 450 mL of solvent A in a graduated cylinder and stir until dissolved. Make up to 1 L with solvent A and filter through a 0.2 μm membrane.

2.4 Solvent C: 1 mM NaCl in Solvent A

Sodium chloride (FW 58.44 J.T. Baker Cat. No. 3624-01), 29.22 g

Solvent A (step 2.2) is added to 500 mL

Combine sodium chloride with approximately 450 mL of solvent A in a graduated cylinder and stir until dissolved. Make up to 500 mL with solvent A and filter through a 0.2 μm membrane.

2.5 Column storage solution: 0.05% sodium azide in solvent B, pH 7.5 ± 0.1

WARNING: Sodium azide is highly toxic and mutagenic. Avoid inhalation of powder and contact with skin (it is readily absorbed through the skin).

Sodium azide (FW 65.01, EM Science 0066884R or equivalent), 2.25 g;

Solvent B (step 2.2), make up to 500 mL

Combine sodium azide with approximately 450 mL of solvent B in a graduated cylinder and stir until dissolved. Make up to 500 mL with solvent B and filter through a 0.22 μm membrane.

2.6 Column and system cleaning solution: 0.1N sodium hydroxide (J T Baker 5636-02), prepared according to the following steps:

1N NaOH 100μL

Purified water 900 μL

Combine the listed chemicals and mix thoroughly.

2.7 Preparation of sample and reference standard buffers

2.8 10% polysorbate 20 stock solution (w/v)

Polysorbate 20 (Polysorbate ^™ 20, Sigma catalog number P7949 or equivalent) 10 g, purified water, make up to 100 mL

Measure polysorbate 20 directly into a tared, graduated cylinder. Avoid contact of the surfactant with the neck of the cylinder. Carefully fill to 100 mL with purified water, avoiding the formation of bubbles. Gently lower a magnetic stir bar into the cylinder. Stir the solution for 15-20 minutes until all the surfactant has dissolved.

2.9 Method Control Preparation Buffer

MAb8 formulation buffer: 20 mM histidine HCl, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0 ± 0.3

L-Histidine hydrochloride, monohydrate (FW 209.6) 2.31g

L-Histidine, free base (FW 155.2) 1.40g

Sucrose (FW 342.3) 41.08g

Polysorbate 20 0.20g

or 10% polysorbate 20 (w/v) stock solution 2.0 mL

Purified water to make up to 1.0L

Combine the listed chemicals with approximately 800 mL of purified water and stir until dissolved. Verify that the pH is 6.0 ± 0.3. If the pH falls outside the specified range, discard and repeat the preparation. Make up the solution to 1.0 L with purified water. Filter through a 0.45 μm membrane.

2.10 5 mg/mL carboxypeptidase B, DFP treated (Roche 103233) or equivalent, approximate activity 150 U/mg

2.11 1mg/mL CpB

5mg/mL Carboxypeptidase B, DFP treated 20μL

Purified water 80 μL

Add exactly 5 mg/mL of Carboxypeptidase B to purified water. For concentrations of purchased Carboxypeptidase B other than 5 mg/mL, adjust the volume to ensure a final concentration of 1 mg/mL. Prepare fresh.

Preparation of method controls, samples, references, and formulation buffer blanks

3.1 Method control (MAb8), nominal concentration: 50 mg/mL

Dilute the method control with solvent A (step 2.2) to a final concentration of approximately 2.0 mg/mL (eg, for a 50 mg/mL method control, combine 40 μL of sample and 960 μL of solvent A).

3.2 Method control blank

Dilute the Method Control Formulation Buffer with Solvent A using the same dilution scheme as in step 3.1.

3.3 Sample and reference standard preparation

Samples and reference standards were diluted with solvent A to 2 mg/mL.

3.4 Sample and Reference Standard Blank Preparation

3.4.1 Using the same dilution scheme as in step 3.4, dilute the product preparation buffer

3.5 Record the dilution on the data sheet.

3.6 Refer to the product specific information and CpB digestion instructions to prepare samples using CpB digestion

3.6.1 Add 1% (w/w) of 1 mg/mL CpB (step 2.12) to the diluted method controls, samples, reference standards, and formulation buffer blanks (e.g., add 20 L of 1 mg/mL CpB to 1000 L of 2.0 mg/mL sample).

3.6.2 Vortex gently to mix and incubate the CpB-treated method controls, samples, reference standards, and formulation buffer blanks at 37 ± 2 °C for 20 ± 2 minutes.

3.6.3 Record the preparation process on the data sheet.

3,7 Transfer the diluted method controls, reference standards, samples, and formulation buffer blanks into appropriate vials for analysis.

3,8HPLC analysis should be completed within 48 hours of sample preparation. Samples should be stored at 2-8°C prior to analysis.

Chromatography conditions

4.1 Common chromatographic conditions for both Water HPLC instruments:

4.1.1 Flow rate: 1.5 mL/min

4.1.2 Automatic sampler temperature: 5±3℃

4.1.3 Column temperature: 40 ± 2 °C

4.1.4UV detection wavelength: 280nm

4.1.5 Injection volume: 25L (about 50g)

4.2 Instrument Setup for Water's Aqcuity H-Class UPLC and Multi-Wavelength or Diode Array Detectors

4.2.1 Zero offset analog output: 5%

4.2.2 Attenuation analog output: 500mAU

4.2.3 Washing environment: Injection with needle wash solution (10% IPA)

10 seconds before injection

20 seconds after injection

4.2.4 Aspiration and dispensing speed: 100 μL/min

4.2.5 Acceleration 2.0 mL/min/0.02 min (100 mL/min/min)

4.2.6 Detector Settings

4.2.6.1 Sampling rate: 1pt/second

4.2.6.2 Filter: Hamming

4.2.6.3 Time constant 1.0

4.2.6.4 Minimum ratio: 0.00 Maximum ratio: 2.00

4.2.6.5 Auto Zero Channel A: (Time 0 and Time 50)

4.2.6.6 Sensitivity: 2.000 AUFS

4.3 Instrument settings for Waters Alliance(e)2695 HPLC with Waters2487 detector

4.3.1 Stroke output: 100μL

4.3.2 Injection needle washing time: extended (10% IPA)

4.3.3 Solvent degassing: Set to "On" mode

4.3.4 Acceleration 10.0 mL/min/0.1 min (100 mL/min/min)

4.3.5 Aspiration and dispensing speed: slow (50 μL/min)

4.3.6 Detector Settings

4.3.6.1 Sampling rate: 1pt/second

4.3.6.2 Filter: Hamming

4.3.6.3 Time constant 1.0

4.3.6.4 Ratio minimum 0.1000

4.3.6.5 Auto-zero channel A at time 0 and time 50

4.3.6.6 Sensitivity: 2.000AU

4.4 Gradient:

Table 13

Time (minutes) %A %B %C Flow rate (mL/min) 0.0 100 0.0 0.0 1.5 3.0 100 0.0 0.0 1.5 37.0 10.0 90.0 0.0 1.5 37.1 0.0 0.0 100 1.5 40.0 0.0 0.0 100 1.5 40.1 100 0.0 0.0 1.5 50.0 100 0.0 0.0 1.5

Instrument preparation

5.1 Follow an appropriate protocol using HPLC

5.2 Prepare the tubing with approximately 20 mL of appropriate solvent, including 10% IPA to prepare the needle wash tubing.

Column cleaning and preparation

6.1 Perform system and column washes by using the following isocratic program: Inject 100 μL of 0.1 N NaOH.

Table 14

Time (minutes) Flow rate (mL/min) %Solvent B %Solvent B 0 1.5 50 50 3 1.5 50 50

6.2 Repeat step 6.1 at least five (5) times.

6.3 Using the isocratic program from step 6.1, make a single injection of 100 μL of solvent A.

6.4 Equilibrate the column at the initial conditions of the gradient program in step 4.4 (100% solvent A at 1.5 mL/min) for approximately 20 minutes or until a stable baseline is observed.

Injection plan

7.1 Preparation: Inject a method control without the CpB digest until a consistent chromatogram is observed for at least two injections. The resolution of the acidic region, main peak, and basic region must be consistent with the representative chromatogram by visual inspection.

7.2 Platform Method Control Without CpB Digestion (Single Injection)

7.3 Preparation Buffer Blank for Method Control

7.4 Reference Standard* (Single Injection)

7.5 samples* (repeated injections)

7.6 Reference Standard* (Single Injection)

7.7 Reference Standard Preparation Buffer Blank* (Single Injection)

7.8 Platform Method Control Without CpB Digest (Single Injection)

*With or without CpB digest, if product is warranted.

Notes: 1) If the preparation buffer is different for the reference standard and samples, inject separate blanks of the reference standard and samples.

2) If more than 15 injections are required between method controls (including the reference standard and the corresponding product preparation buffer blank), insert a method control injection between every 15 injections. In the system suitability section of the experimental data sheet, report only the method control injections inserted between the samples being reported.

3) The reference standard is considered a sample and is not used to assess the system suitability of the test procedure.

Column Shutdown and Storage

Store the column (step 2.4) by flushing it with at least 30 mL of column storage solution.

System suitability

Note: For the method control, determine the integration endpoint by overlaying the method control curve with a blank of the method control preparation buffer. Extend the overlay curve and determine the integration endpoint by comparing the blank to the method control curve.

9.1 Integrate all peaks attributable to the protein. Do not include any peaks present in the method control formulation buffer blank chromatogram unless the corresponding peak in the blank is less than 1% of the peak in the PMC.

9.2 Visually verify the consistency of the chromatograms of the intervening method control injections with each other and with the representative chromatogram. All specified peaks in the representative chromatogram must be present.

Note: The curve for a given peak may differ slightly in peak shape from the example curve due to column and instrument variability.

9.3 Calculate the percentage of the main peak, acidic region, and basic region for each bracketing method control injection.

9.4 System Applicability Scope

Table 15. Acceptable ranges for system suitability of non-CpB treated method controls

acidic area Main Peak alkaline region Acceptable range of peak area% 15.5 and 17.7 55.5 and 61.4 21.3 and 28.6

9.5 Record the results on the System Suitability Data Sheet.

Data Analysis

10.1 Visually compare the curves to identify the peaks in the sample chromatogram and the reference standard chromatogram.

10.2 Integrate all peaks attributable to the protein. Do not include any peaks present in the blank chromatogram of the product preparation buffer, unless the corresponding peak in the blank is less than 1% of the peak in the PMC. Note: To determine the integration endpoint, overlay the sample and reference standard curves with the product preparation buffer blank. Extend the overlay curve and determine the integration endpoint by comparing the product preparation buffer blank with the sample and reference standard curves.

10.3 Analyze the amount of each sample injected and the amount of the reference standard injected to calculate the peak area % of the main peak, acidic region and basic region.

Claims (86)

1. A method for determining optimal ion exchange chromatography separation conditions for analyzing a variety of compositions, wherein each composition comprises a polypeptide along with one or more contaminants, the method comprising: a) Based on the amino acid composition of the peptides from two or more compositions, plot the net charge versus pH curve at a selected temperature, and b) By determining the second derivative of the curve in step a), determine the inflection point of the net charge-to-pH curve at or near neutral pH. The optimal ion exchange chromatography separation conditions are the pH values at approximately the common inflection point of the polypeptides in one or more compositions. 2. The method according to claim 1, wherein if the net charge is positive at the inflection point, the cation exchange material is used for ion exchange chromatography. 3. The method according to claim 2, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material. 4. The method according to claim 1, wherein if the net charge is negative at the inflection point, the anion exchange material is used for chromatography. 5. The method according to claim 4, wherein the anion exchange chromatography material is a quaternary ammonium chromatography material or a tertiary ammonium chromatography material. 6. The method of claim 1, wherein the mixed-mode chromatography material is used for chromatography. 7. The method according to claim 6, wherein the mixed-mode ion exchange material is a mixture of sulfonated or carboxylated chromatographic material and quaternary or tertiary amine chromatographic material packed sequentially. 8. The method according to any one of claims 1-7, further comprising: c) For peptides composed of two or more components, determine the inflection point of the net charge-to-pH curve and the change in pH as a function of temperature (dIP/dT). d) Select a buffer for chromatography in which the acid dissociation constant of the buffer changes with temperature (dpKa/dT) in a manner that is substantially the same as that of the peptide (dIP/dT). 9. The method of claim 8, wherein the buffer provides effective buffering capacity at the inflection point pH. 10. The method according to any one of claims 1-7, wherein the dIP/dT of the polypeptide of one or more compositions is about -0.02 pH units. 11. The method according to any one of claims 1-7, wherein the temperature change is from about 20°C to about 70°C. 12. The method according to any one of claims 1-7, wherein the temperature change is from about 20°C to about 50°C. 13. The method according to any one of claims 1-7, wherein dpKa/dT = dIP/dT ± 50%. 14. The method according to any one of claims 1-7, wherein the net charge in the buffer selected by the polypeptide in step d) varies by less than 0.5 above 30°C. 15. The method according to any one of claims 1-7, wherein the buffer selected in step d) is used in the chromatography at a concentration ranging from about 5 mM to about 250 mM. 16. The method according to any one of claims 1-7, wherein the buffer composition further comprises salt. 17. _The method according to claim 16, wherein _the salt is NaCl, KCl, ( _NH4 ) _2SO4 or _Na2SO4 . 18. The method of claim 17, wherein the concentration of the salt is from about 1 mM to about 1 M. 19. The method according to any one of claims 1-7, wherein the polypeptide is an antibody or an immunoadhesin or a fragment thereof. 20. The method according to any one of claims 1-7, wherein the polypeptide is a monoclonal antibody or a fragment thereof. 21. The method of claim 19, wherein the antibody is a human antibody. 22. The method of claim 19, wherein the antibody is a humanized antibody. 23. The method of claim 19, wherein the antibody is a chimeric antibody. 24. The method of claim 19, wherein the antibody is an antibody fragment. 25. The method according to any one of claims 1-7, wherein the contaminant is a variant of a polypeptide. 26. The method according to any one of claims 1-7, wherein the contaminant is a degradation product of the polypeptide. 27. The method according to any one of claims 1-7, wherein the contaminant is a charge variant of the polypeptide. 28. A method for determining optimal ion exchange chromatography separation conditions for analyzing compositions comprising peptides along with one or more contaminants, comprising: a) Based on the amino acid composition of the peptide, plot the net charge versus pH curve at a selected temperature, and b) By determining the second derivative of the curve in step a), determine the inflection point of the net charge-to-pH curve at or near neutral pH. The optimal ion exchange chromatography separation conditions are the pH values around the inflection point of the peptide. 29. The method of claim 28, wherein if the net charge is positive at the inflection point, the cation exchange material is used for ion exchange chromatography. 30. The method of claim 29, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material. 31. The method of claim 28, wherein if the net charge is negative at the inflection point, the anion exchange material is used for chromatography. 32. The method according to claim 31, wherein the anion exchange chromatography material is a quaternary ammonium chromatography material or a tertiary ammonium chromatography material. 33. The method of claim 28, wherein the mixed-mode chromatography material is used for chromatography. 34. The method of claim 33, wherein the mixed-mode ion exchange material is a mixture of sulfonated or carboxylated chromatographic material and quaternary or tertiary amine chromatographic material packed sequentially. 35. The method according to any one of claims 28-34, further comprising: c) For peptides, determine the inflection point of the net charge-to-pH curve and the change in pH with temperature (dIP/dT). d) Select a buffer for chromatography in which the acid dissociation constant of the buffer changes with temperature (dpKa/dT) in a manner that is substantially the same as that of the peptide (dIP/dT). 36. The method of claim 35, wherein the buffer provides effective buffering capacity at the inflection point pH. 37. The method according to any one of claims 28-34, wherein the dIP/dT of the polypeptide of one or more compositions is about -0.02 pH units. 38. The method according to any one of claims 28-34, wherein the temperature change is from about 20°C to about 70°C. 39. The method according to any one of claims 28-34, wherein the temperature change is from about 20°C to about 50°C. 40. The method according to any one of claims 28-34, wherein dIP/dT = dpKa/dT ± 50%. 41. The method according to any one of claims 28-34, wherein the net charge in the buffer selected by the polypeptide in step d) varies by less than 0.5 above 30°C. 42. The method according to any one of claims 28-34, wherein the buffer selected in step d) is used in the chromatography at a concentration ranging from about 5 mM to about 50 mM. 43. The method according to any one of claims 28-34, wherein the buffer composition further comprises salt. 44. _The method according to claim 43, wherein _the salt is NaCl, KCl, ( _NH4 ) _2SO4 or _Na2SO4 . 45. The method of claim 44, wherein the concentration of the salt is from about 10 mM to about 1 M. 46. The method according to any one of claims 28-34, wherein the polypeptide is an antibody or an immunoadhesin or a fragment thereof. 47. The method according to any one of claims 28-34, wherein the polypeptide is a monoclonal antibody or a fragment thereof. 48. The method of claim 46, wherein the antibody is a human antibody. 49. The method of claim 46, wherein the antibody is a humanized antibody. 50. The method of claim 46, wherein the antibody is a chimeric antibody. 51. The method according to any one of claims 28-34, wherein the antibody is an antibody fragment. 52. The method according to any one of claims 28-34, wherein the contaminant is a variant of a polypeptide. 53. The method according to any one of claims 28-34, wherein the contaminant is a degradation product of the polypeptide. 54. The method according to any one of claims 28-34, wherein the contaminant is a charge variant of the polypeptide. 55. A method for analyzing a composition, wherein the composition comprises a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising: a) Determine the optimal pH and temperature ion exchange separation conditions for a variety of compositions, each composition comprising a target peptide and one or more contaminants, using the method described in claim 1. b) Using a loading buffer to bind peptides and one or more contaminants from the composition to an ion exchange chromatography material, wherein the loading buffer contains a buffer determined by the method of any one of claims 8-15; c) Using a gradient of elution buffer, peptides and one or more contaminants are eluted from ion-exchange chromatography material, wherein the elution buffer contains a buffer and a salt, wherein the salt concentration increases over time in the gradient, and wherein the peptides and one or more contaminants are separated by the gradient; and d) Detect peptides and one or more contaminants. 56. A method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising: a) Using a loading buffer, peptides and one or more contaminants are bound to ion-exchange chromatography material, wherein the loading buffer contains a buffering agent, and the pH and temperature of the chromatography have been optimized for multiple target peptides in the following manner. i) Plot a net charge-to-pH curve at a selected temperature, where the curve is based on the amino acid composition of the peptides for two or more target peptides, and ii) Determine the inflection point of the net charge versus pH curve by determining the second derivative of the curve from step i). The optimal ion exchange chromatography conditions are the pH values at the common inflection point of two or more target peptides. b) Using a gradient of elution buffer, peptides and one or more contaminants are eluted from ion-exchange chromatography material, wherein the elution buffer contains buffer and salt. In this process, peptides and one or more contaminants are separated by a gradient; and c) Detection of peptides and one or more contaminants. 57. The method of claim 56, wherein the selected temperature is the ambient temperature. 58. The method of claim 56 or 57, wherein the buffer is determined by: a) For two or more target peptides, determine the inflection point of the net charge-to-pH curve and the change in pH with temperature (dIP/dT). b) Select a buffer in which the acid dissociation constant (dpKa/dT) changes with temperature substantially the same as the dIP/dT of one or more target peptides having a common inflection point. 59. The method of claim 58, wherein the buffer provides effective buffering capacity at the inflection point pH. 60. A method for analyzing a composition comprising a polypeptide and one or more contaminants, wherein the method effectively separates the polypeptide from the contaminants, the method comprising: a) Determine the optimal pH and temperature ion exchange separation conditions for various compositions using the method described in claim 1. b) Using a loading buffer to bind peptides and one or more contaminants to an ion-exchange chromatography material, wherein the loading buffer contains a buffer determined by the method of any one of claims 8-15, and wherein the pH and temperature of the chromatography have been optimized for a variety of target peptides; c) Using a gradient of elution buffer, peptides and one or more contaminants are eluted from ion-exchange chromatography material, wherein the elution buffer contains buffer and salt. In this process, peptides and one or more contaminants are separated by a gradient; and d) Detect peptides and one or more contaminants. 61. The method of claim 60, wherein the buffer is N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) or (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). 62. The method according to any one of claims 55-61, wherein the concentration of the buffer is from about 5 mM to about 20 mM. 63. The method according to any one of claims 55-61, wherein the pH of the buffer is in the temperature range of about 6.5 to about 8.5 from about 20°C to about 70°C. 64. The method according to any one of claims 55-61, wherein the pH of the buffer is in the temperature range of about 6.5 to about 8.5 from about 20°C to about 50°C. 65. The method according to any one of claims 55-61, wherein the pH of the buffer and the peptide at the inflection point is about 7.8 at about 22°C, about 7.5 at about 37°C, or about 7.2 at about 50°C. 66. The method according to any one of claims 55-61, wherein the salt gradient is a linear gradient. 67. The method according to any one of claims 55-61, wherein the salt gradient is a step gradient. 68. The method according to any one of claims 55-61, wherein the salt gradient is a NaCl gradient, a KCl gradient, _a ( _NH4 ) _2SO4 gradient, or a _{Na2SO4 gradient} . 69. The method according to any one of claims 55-61, wherein the salt concentration in the gradient increases from about 0 mM to about 1 M. 70. The method of claim 69, wherein the salt concentration increases from about 0 mM to about 100 mM over about 100 minutes. 71. The method of claim 69, wherein the salt concentration increases from about 0 mM to about 80 mM over about 40 minutes. 72. The method according to any one of claims 55-61, wherein the polypeptide is an antibody or an immunoadhesin or a fragment thereof. 73. The method according to any one of claims 55-61, wherein the polypeptide is a monoclonal antibody or a fragment thereof. 74. The method of claim 72, wherein the antibody is a human antibody. 75. The method of claim 72, wherein the antibody is a humanized antibody. 76. The method of claim 72, wherein the antibody is a chimeric antibody. 77. The method of claim 72, wherein the antibody is an antibody fragment. 78. The method according to any one of claims 55-61, wherein the contaminant is a variant of a polypeptide. 79. The method according to any one of claims 55-61, wherein the contaminant is a degradation product of the polypeptide. 80. The method according to any one of claims 55-61, wherein the contaminant is a charge variant of the polypeptide. 81. The method according to any one of claims 55-61, wherein the chromatographic material is a cation exchange chromatographic material. 82. The method according to claim 81, wherein the cation exchange chromatography material is a sulfonated chromatography material or a carboxylated chromatography material. 83. A method for analyzing multiple polypeptide compositions, wherein each polypeptide composition comprises a polypeptide and one or more charge variants of the polypeptide, wherein the method effectively separates the polypeptide from its charge variants; For each polypeptide composition, the method includes, a) Use a loading buffer at a flow rate of approximately 1 mL/min to bind the peptide and one or more charge variants to the ion exchange chromatography material, wherein the loading buffer is contained in a 10 mM HEPES buffer at approximately pH 7.6 at approximately 40 °C. b) Using a gradient of elution buffer, peptides and charge variant contaminants are eluted from ion exchange chromatography material, wherein the elution buffer contains 10 mM HEPES buffer and NaCl at approximately pH 7.6, wherein the concentration of NaCl in the gradient increases from approximately 0 mM to approximately 80 mM over approximately 40 minutes, wherein the peptides and their charge variants are separated by the gradient; and c) Detect peptides and one or more charge variants. 84. The method of claim 83, wherein the plurality of polypeptide compositions comprises different polypeptides. 85. The method according to claim 83 or 84, wherein the plurality of polypeptide compositions comprises polypeptides having different pIs. 86. The method according to claim 83 or 84, wherein the polypeptide composition is an antibody composition.