WO2015083853A1 - Method for preparing antibody through adjustment of amount of heterogeneous antibodies - Google Patents

Abstract

The present invention relates to a method for preparing a high purity and high quality population of antibodies by adjusting, to a desired level, the amount of heterogeneous antibodies in a population of antibodies, a population of antibodies prepared by the method, a method for adjusting the amount of heterogeneous antibodies in the population of antibodies, and a method for determining a pH settling time to adjust the amount of heterogeneous antibodies. By using the method of the present invention, the amount of heterogeneous antibodies in the population of antibodies is adjusted to a desired amount, thereby consistently preparing the population of antibodies having ensured high quality or equivalency.

Description

Antibody Production Method Through Control of Content of Isomer Antibodies

The present invention provides a method for producing an antibody, the method for producing a high purity and high quality antibody population by adjusting the content of the isomeric antibody in the population of antibodies (population of antibodies) to the target content, the antibody population prepared by the above method, the antibody population The present invention relates to a method for controlling the content of isomeric antibodies in a body and a method for determining the pH settling time for controlling the content of isomeric antibodies.

In antibody purification, maintaining quality consistency with impurity removal is an important issue. The antibody has a structure in which two heavy chains and two light chains are disulfide bonds, and glycosylation is performed near the Fc of the heavy chain. Antibodies produced using CHO cells as a host include various isomeric antibodies (Hongcheng Liu, Georgeen Gaza-Bulseco, Journal of Chromatography B, 837 (2006) 35-43), and isomeric antibodies are deamidation. Most of the structure is modified by several amino acids (oxidation, etc.) (Isamu Terashima, Akiko Koga, Analytical Biochemistry 368 (2007) 49-60), especially the complementarity determining region (CDR), the antibody antigen binding site It is reported that demineralized isomer antibodies may affect the activity by decreasing the binding capacity of the antigen (Reed J. Harrisa, Bruce Kabakoffb Journal of Chromatography B, 752 (2001) 233-245).

As the isomeric antibody, isparagine is an amino acid deamined as an aspatate (Boxu Yan, Sean Steen, Journal of Pharmaceutical Sciences, Vol. 98, No. 10, October 2009), Isomers in which amino acid methionine is oxidized to become methionine sulfate (Chris Chumsae, Georgeen Gaza-Bulseco, Journal of Chromatography B, 850 (2007) 285-294). In addition, when glutamate is present at the N-terminus of the heavy chain, the glutamate may form a pentagonal ring structure to be transformed into pyruglutamate (William E. Werner, Sylvia Wu, Analytical Biochemistry 342 (2005) 120-125). Since these isomeric antibodies affect the biological activity of the antibody, it is necessary to control the amount of the isomeric antibody to a certain limit in the production of the antibody.

Isomer antibodies vary in rate of deamination depending on pH and temperature, and are generally studied for conditions under which deamination can be minimized in terms of stability. However, in the biosimilar aspect, if the content of the isomeric antibody is different from the reference drug, the quality may be different in terms of efficacy, and thus may be a problem in terms of equivalence evaluation. Therefore, the development of a method of maintaining a constant content of isomeric antibodies in the final purification product is greatly demanded in the biosimilar antibody pharmaceutical market.

In the manufacture of antibody pharmaceuticals, under the background of the great demand for the development of a method of maintaining a constant content of isomeric antibodies in a purified product, the present inventors have controlled the content of isomeric antibodies in biosimilar antibody pharmaceuticals to increase the content of isomeric antibodies. Efforts have been made to develop a method of maintaining a constant method, and it has been confirmed that by allowing the reaction to stand at an appropriate pH, constant temperature and time, the isomeric antibody can be controlled at a desired ratio, thereby selectively increasing the specific isomeric antibody and controlling it. The present invention was completed by confirming that the pattern of isomeric antibodies similar to drugs, specifically high quality or equivalence.

One object of the present invention is to (a) selecting a pH and time to prepare an isomeric antibody of a desired content; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a), wherein the antibody population comprises a desired amount of isoform. It is to provide a method for producing.

Another object of the present invention is to provide an antibody population comprising the desired amount of isomeric antibodies prepared by the above method.

Another object of the present invention is to select a pH and time to prepare a desired amount of isomeric antibody; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a). To provide.

Another object of the present invention is to select a pH at which (a) to prepare isomeric antibodies of a desired content; And (b) allowing the sample containing the mixed solution of the antibody to remain within the pH selected in step (a), to control the content of the isoform in the antibody population or to a desired content. In the manufacturing method of the antibody population containing an isomeric antibody, it provides the method of determining pH settling time using the hourly rate of increase of specific main acidic peak%.

Using the method for producing an antibody according to the present invention, the content of isomeric antibodies can be controlled to consistently produce a high quality antibody population of interest. In addition, in terms of the development of biosimilars, the antibody according to the present invention can be manufactured by controlling the content of the isomeric antibody to improve the equivalence with the reference drug, and can shorten the existing process time. This can be high.

1 is a diagram showing a pattern change of the isomeric antibody over time by the pH rise. 1A is a diagram showing a pattern change of isomeric antibodies after 4 hours of pH maturation, and FIG. 1B is 24 hours of pH maturation.

Figure 2 is a view showing the range of a typical acidic, main, basic portion on CEX HPLC.

Figure 3 is a diagram showing the pattern change of the isomeric antibody at 12 hours pH maturation. 3A shows CEX HPLC Full and FIG. 3B shows CEX HPLC expansion, respectively.

Figure 4 is a diagram showing the pattern change of the isomeric antibody at 24 hours pH maturation. 4A shows CEX HPLC Full, and FIG. 4B shows CEX HPLC expansion.

5 is a diagram comparing the content of each portion during pH maturation (12hr vs 24hr). 5A shows a main acidic peak (AM)%, FIG. 5B shows an acidic portion%, FIG. 5C shows a main portion%, and FIG. 5D shows a basic portion%.

Figure 6 is a diagram showing the change in AM (main acidic peak) and acidic portion content at pH 7.5 maturation in the culture supernatant (3, 6, 9, 24hr). 6A shows main acidic peaks (AMs), and FIG. 6B shows acidic portion%.

Figure 7 is a diagram showing the pattern change of the isomeric antibody during pH maturation in the culture supernatant (temperature: 25 ℃, 30 ℃, 37 ℃, time: 24hr). 7A shows CEX HPLC Full and FIG. 7B shows CEX HPLC expansion, respectively.

Figure 8 is a schematic diagram of the process flow and pH maturation insertion step of the present inventors Trastuzumab purification process.

9 is a view showing a comparison of the content of each portion during pH maturation in CEX and VI step (time: 6, 11, 24hr, temperature: 4 ℃, 25 ℃). 9A shows main acidic peak%, FIG. 9B shows acidic portion%, FIG. 9C shows main portion%, and FIG. 9D shows Basic Portion%.

10 is a diagram showing the pattern change of the isomeric antibody during pH maturation in CEX and VI (temperature: 25 ℃, time: 11hr, 24hr).

11 is a diagram comparing the content of each portion during pH maturation in the HIC step (time: 6, 9, 24hr, temperature: 4 ℃, 25 ℃). 11A shows the main acidic peak (AM)%, FIG. 11B shows the acidic portion%, FIG. 11C shows the main portion%, and FIG. 11D shows the Basic Portion%.

12 is a view showing a pattern change of the isomeric antibody during pH maturation in the HIC step (temperature: 4 ℃, 25 ℃, time: 24hr).

Figure 13 is a diagram showing the content comparison for each portion during pH maturation in UF / DF1 step (time: 6, 24hr, temperature: 4 ℃, 25 ℃). FIG. 13A shows the main acidic peak (AM)%, FIG. 13B shows the acidic portion%, FIG. 13C shows the main portion%, and FIG. 13D shows the Basic Portion%.

14 is a view showing a pattern change of the isomeric antibody during pH maturation in UF / DF1 step (temperature: 25 ℃, 4 ℃, time: 24hr).

15 is a diagram showing the peak containing AM in CEX HPLC.

16 is a diagram showing AM growth rate verification by Plot between AM% and maturation time.

Figure 17 is a graph confirming the content control of isomeric antibodies in the production of Trastuzumab 1000ℓ. 17a shows CEX, UF / DF2, and reference overlap (No overlap), and FIG. 17B shows CEX, UF / DF2, and reference comparison (overlap).

In one aspect, the present invention provides a method for consistently producing a high-quality or equivalence antibody population, by adjusting the pH, temperature, time, etc. in the culture or purified solution to the desired content of the isomeric antibody Provided are methods for producing an antibody population comprising.

Specifically, the present invention comprises the steps of (a) selecting the pH and time to prepare the isomeric antibody of the desired content; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a), wherein the antibody population comprises a desired amount of isoform. It is possible to provide a method for producing. In the present invention, the term "pH settling" may be used interchangeably with pH maturation.

Antibody products prepared through host cells include several isomeric antibodies in addition to the main active antibody. The isomeric antibody is an antibody in which some amino acids in the antibody are modified by deamine or oxidation, and the isomeric antibodies differ in biological activity. Antibody products expressed through host cells may produce inconsistent amounts of such isomeric antibodies. In particular, in the case of antibody biosimilars, preparation of a quality similar to that of a reference drug is important. Therefore, a process for producing an antibody from host cells and then controlling the content of the isomeric antibody is required. Therefore, the present invention has been developed a method for controlling the content of isomeric antibodies, which surprisingly only raises a specific peak of the isomeric antibodies when the culture or purified liquid used for the production of the antibody is left at a certain pH for a certain time. Based on this, there is provided a method capable of effectively producing a high quality or equivalency antibody population comprising a desired ratio of isomeric antibodies.

As used herein, the term "a population of antibodies" refers to an antibody group comprising a main active antibody and an isomeric antibody. For the purposes of the present invention, the antibody population is intended for a main active antibody and an isomeric antibody. It means the antibody group included in the ratio to do. The antibody population includes only one type of antibody, or includes a group of antibodies containing both main active and isomeric antibodies. For the purposes of the present invention said antibody population preferably means an antibody population comprising a desired amount of isomeric antibodies. Preferably, the antibody population has a difference in quality such as efficacy when the content of the isomeric antibody, which is considered to be important in the biosimilar antibody medicine for the purpose of the present invention, is different from the reference drug, thereby causing problems in evaluating equivalence. By eliminating the possible disadvantages, it can mean an antibody population comprising a content of isomeric antibodies within the same or similar range as the biosimilar counterpart.

In the present invention, the term "control" means a drug that is a subject of replication of the biosimilar drug, but is not limited thereto and may mean a drug of a target that is intended to ensure equivalence.

In particular, when the method of the present invention is applied to the production of antibody biosimilars, antibody populations comprising the main active antibody and the isomeric antibody can be prepared in the same or corresponding composition as the reference drug.

As used herein, the term “antibody” refers to a substance produced by stimulation of an antigen in the immune system, and specifically binds to a specific antigen, causing a substance to float on lymph and blood to generate an antigen-antibody reaction. For the purposes of the present invention, the antibody is one of proteins for high quality purification, and can be efficiently prepared and purified by the method according to the present invention.

Since the antibody generally has a higher isoelectric point than other proteins, the primary supernatant can be purified with relatively high purity by adsorbing the culture supernatant on the column using initial cation exchange chromatography. The isoelectric point (pI) is an average effective charge on the surface of the protein molecule, that is, a pH value at which the potential of the electrical double layer of the protein molecule becomes zero, and the effective charge is caused by the dissociation of the groups of the protein and the equal number of positive and negative groups. This means that the point becomes zero. The antibody to be purified in the present invention is not limited thereto, but may preferably be an antibody having an isoelectric point of 7 to 11, more preferably 8 to 10. In addition, the antibody of the present invention is not limited thereto, but may preferably include all therapeutic antibodies commonly used in the art, and more preferably targets Human Epidermal Growth Factor Receptor 2 (HER-2). The antibody may be trastuzumab or pertuzumab, and most preferably trastuzumab. Trastuzumab, also known as Herceptin, is a humanized antibody against HER2 developed by Genentech in the United States, which is known as an antibody therapeutic against HER2 / neu mainly expressed in breast cancer cells.

As used herein, the term "mainly active antibody" is a major component included in the antibody population of the present invention, wherein some amino acids in the antibody are modified by deamine or oxidation, so that the biological activity is not lowered, That is, it means an antibody that is not an acidic or basic isomeric antibody. The main active antibody is the most important component for controlling the quality of the desired antibody population, the antibody with the highest biological activity among the components of the antibody.

As used herein, the term “isomer antibody” refers to an antibody in which some amino acids of the main active antibody are modified by deamine or oxidation, and include acidic isomers and basic isomeric antibodies. Examples include isomers in which asparagine is deamined in amino acids to form aspatate, and isomers in which methionine is oxidized to become methionine sulfate. In addition, when glutamate is present at the N-terminus of the heavy chain, the glutamate forms an pentagonal ring structure and includes an isomeric antibody modified with pyruglutamate. When isomeric antibodies are included in the host cell culture at a high rate when the antibody is produced in host cells, such as CHO cells, some of the isomeric antibodies must be removed through a process such as chromatography to be included in the antibody population at the desired rate. . Preferably, the isomeric antibody may be an acidic isomeric antibody, but is not limited thereto. Since there are many isomeric antibodies in the antibody product expressed through the host cell, it is important to demonstrate homogeneity to make the quality most similar to the reference drug to prepare the antibody biosimilar. The isomeric antibodies are a modified form of several amino acids of the main active antibody, with slight differences in charge between the main active antibody and the acidic isomeric antibody.

In addition, the isomeric antibodies are modified by deamine or oxidation of some amino acids in the antibody, it is known that there is a difference in biological activity for each isomeric antibody, it is consistent quality to bring a constant content distribution of the isomeric antibodies Is important to maintain.

Therefore, in a host cell into which a vector containing a polynucleotide encoding an antibody is introduced, in order to prepare a high quality antibody population, the above-mentioned isomer antibody should be appropriately removed so that the main active antibody and isomer antibody are included in a desired amount. There is a need to do it. Accordingly, the present invention has been developed a method for producing an antibody population, which can adjust the content of the isomeric antibody according to the desired content. For example, when preparing an antibody drug such as trastuzumab, the content of the isomeric antibodies may be relatively lower than that of the control drug depending on the culture conditions. As a result of the pH fixation process to raise the acid isomer antibody to the same quality as the reference drug, the specific peak of the isomer antibody, specifically, the main acidic peak (AM), is specifically raised when standing at a high pH for a certain time. It was confirmed that the rate of increase of peaks other than, was minimized (FIGS. 1A and 1B). These results suggest that antibody populations containing the desired amount of isomeric antibodies can be prepared upon standing at a particular pH.

The mixed solution of the antibody of step (a) may be a culture solution for producing an antibody or a purification solution obtained in various purification steps, and the culture solution may be a culture solution containing cells or a culture supernatant from which cells have been removed.

The cell removal method may be a method commonly used in the art, but is not limited thereto. Preferably, a filter, more preferably a filtration filter or an ultrafiltration may be used.

As used herein, the term “desired content of isomeric antibody” means a desired amount of isomeric antibody, depending on the antibody applied to the method of the invention. For the purposes of the present invention, this may mean an antibody that is the same or similar to the reference in the manufacture of the biosimilar antibody drug product, for example, within the range of ± 15% of the content of isomeric antibody in the reference drug, ± 10% It may be in the range, and based on the AM (main acidic peak)%, it may be in the range of ± 17%, within the range of ± 20% and AM of the reference drug, but is not limited thereto, and the desired content It can be variously changed according to the isomeric antibody.

The pH used in the stationary may be pH 6.0 or more pH 9.0 or less, or may be pH 7.0 or more pH 9.0 or less, preferably pH 7.5 or more pH 9.0 or less, or pH 7.0 or more pH 7.5 or less, or pH 8.0 or more. The pH may be 8.2 or less, but is not limited thereto, and the pH used for standing may be selected according to the content of the desired isomeric antibody.

The temperature used for standing within the selected pH may be 4 ° C to which the culture or purified liquid is exposed in various purification steps, preferably 4 ° C or more and 40 ° C or less, more preferably 15 ° C at room temperature. The temperature may be 30 ° C. or lower, but is not limited thereto. If the pH is left at room temperature, there is no need to raise or lower the temperature, which may be advantageous in terms of process.

According to one embodiment of the invention, it was confirmed that AM% increases with each increase in temperature, and furthermore, it was confirmed that AM% increased specifically during pH standing at room temperature (FIGS. 6A and 6B, 7A and 7b).

In addition, the pH settling time may be 1 hour or more and 48 hours or less, preferably 24 hours or less, but is not limited thereto and may be performed by a time selected according to conditions.

The pH and time selection step of step (a) may be performed by the following method.

Specifically, (i) after raising the pH used in the stationary reaction for the same time to obtain AM rate of increase by the following formula at each pH, AM% in the desired AM% range is selected as AM% after the pH stationary, Selecting a pH and AM rise rate to be used in the process; And

[AM Rising Rate Calculation Formula]

Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

(ii) using the AM rate of rise to determine the settling time according to the following equation:

[Political time calculation formula]

pH Settling Time (hr) = (Target AM%-AM% Before pH Settling) ÷ AM Ascent Rate Selected in Step (i) above.

Can be performed by

Increasing the pH at the same time may be variously determined according to experimental conditions. For example, the pH may be increased to 0.01, 0.05, 0.1, 0.5, etc., but is not limited thereto.

After settling at a predetermined pH at various intervals for each sample, the rate of AM rise of each sample is obtained, and then the AM% within the desired AM% range is selected as AM% after pH settling. The rate of increase can be selected. A detailed method thereof has been described in more detail in Example 4.1, and the method thereof may be variously changed according to conditions.

The "desired AM%" may be used interchangeably with the "purpose AM%" in the present invention.

According to one embodiment of the present invention, according to the isomeric antibody content pattern of the initial sample, it was confirmed that by selecting the appropriate pH and the appropriate time can adjust the content of the isomeric antibody, moreover, based on the AM% content It was confirmed that the settling time and pH can be determined when setting.

The pH settling step is to adjust the content of the isomeric antibody in the antibody mixture to the desired content, (i) the mixture of the antibody is purified using cation exchange chromatography (Cation Exchange Chromatography, CEX); (ii) the mixed solution of the antibody was purified by the Virus Inactivation (VI) step; (iii) the mixture of antibodies was purified using Hydrophobic Interaction Chromatography (HIC); (iv) the mixed solution of the antibody was purified by ultrafiltration (Ultrafiltation and Diafiltration, UF / DF); Or (v) a mixture of antibodies can be applied to a variety of antibody purification or preparation steps, such as those purified using Anion Exchange Chromatography, and can be carried out in one or more of each of the above steps. For example, it may be carried out in two or more steps, three or more steps, four or more steps, or all five steps, wherein each step is not a sequential step and a combination of each step depending on the type of antibody population to be produced. Can be selected. In addition, the pH settling step can be performed on the eluate purified in each step.

Preferably, the pH settling step may be performed after the mixed solution of the antibody is purified by the virus inactivation step.

As used herein, the term “cationic exchange chromatography” refers to a column filled with a cation exchange resin, and may be subjected to cation exchange chromatography to remove isomeric antibodies and impurities, preferably host cell proteins. The cation exchange chromatography is a synthetic resin that serves to exchange cations in an aqueous solution with its own cation. Since antibodies have high isoelectric point, they have a cation in a pH buffer below the isoelectric point value. Therefore, the quality of the antibody population can be improved by using cation exchange chromatography capable of adsorbing the cationized antibody. The cation exchange chromatography may be used that is commonly used in the art, but is not limited to this may be preferably a column having a functional group of COO ^- or SO ₃ , more preferably carboxymethyl (CM ), Fructogel, sulfoethyl (SE), sulfopropyl (SP), phosphate (P) or sulfonate (S) and the like, and more preferably carboxymethyl sepharose (CM sepharose). ) Or fructogel COO ⁻ may be used.

As used herein, the term "virus inactivation" includes making a virus contained in a culture or purified liquid non-functional or removing the virus from the culture or purified liquid. Methods for making the virus nonfunctional or removing the virus include a thermal inactivation, pH inactivation, or chemical inactivation method, and the like, but preferably, a pH inactivation method is not limited thereto. The pH inactivation method is a method in which the virus is treated at a pH such that the non-functionality is sufficient, and the method of pH inactivation includes a low pH virus inactivation method. The antibody eluate eluted in the previous chromatography step may be carried out by titration at a pH in the range of 3.0 to 4.0, preferably at pH 3.8, but is not limited thereto.

As used herein, the term "hydrophobic reaction chromatography" means a column filled with a hydrophobic interaction resin, and in this step, a column capable of removing impurities, preferably a host cell protein, may be subjected to hydrophobic interaction chromatography. it means. Proteins are hydrophilic in general but have hydrophobic regions with hydrophilicity, and the hydrophobic nature of these regions is not expressed under strong electrostatic interaction conditions, but the relative weakness of the electrostatic interactions is increased by increasing the ionic strength or dielectric constant of the solvent. It is characterized by strong expression. Here, hydrophobic ligands (long hydrocarbon chains or aromatic rings) are introduced into hydrophilic chromatography substrates (agarose gel-pies, organic polymer supports, etc.) to equilibrate with a strong salt concentration to adsorb various proteins. Lowering it elutes depending on the nature of the protein and can be separated. That is, when a hydrophobic environment is provided using salts, the difference in the strength adsorbed to a specific column is caused by the difference in the hydrophobicity of each protein. By using this principle, the host cell protein using the hydrophobic interaction column can be removed. have.

The hydrophobic interaction resin may be used that is commonly used in the art, but is not limited thereto, and preferably, phenyl column, butyl column, phenyl sepharose or fructogel EMD phenyl column may be used. More preferably, phenyl sepharose may be used.

"Ultrafiltration" in the present invention may be a step for buffer replacement or concentration in the antibody mixture.

As used herein, the term "anion exchange chromatography" refers to a column filled with an anion exchange resin, and in this step, a column capable of removing an impurity, preferably a host cell protein, is performed by performing anion exchange chromatography. This is not restrictive. The anion exchange resin is a synthetic resin that serves to exchange its own anion with a specific anion in an aqueous solution, the anion exchange column may adsorb a protein having an anion above the isoelectric point. In the case of the antibody, since the isoelectric point is high, when the neutral pH buffer is used, the antibody does not adhere to the anion exchange resin, but the impurities including the host cell protein may be adsorbed and removed by the anion exchange resin because the isoelectric point is low. Can be used for the preparation of high purity antibody populations.

The anion exchange resin may be used that is commonly used in the art, but is not limited thereto, preferably Q sepharose, quaternary aminoethyl or quaternary amine (Q) and the like. More preferably, Q Fast Flow may be used.

In addition, since the anion exchange column is an efficient column for removing endotoxin as well as the host cell protein, the desired antibody population with high purity can be purified by removing endotoxin along with the host cell protein in the final purification step.

Preferably, the method for producing an antibody population comprising the desired content of isomeric antibodies comprises the steps of: (a) subjecting a sample comprising a mixture of antibodies to Cation Exchange Chromatography (CEX) for purification; (b) virus inactivation (VI) of the mixed solution of the antibody purified in step (a) to an acidic pH; (c) purifying the mixed solution of the virus-inactivated antibody of step (b) by applying hydrophobic reaction chromatography (HIC); (d) purifying the antibody mixture purified in step (c) by primary ultrafiltration (1st Ultrafiltation and Diafiltration, UF / DF I); And (e) subjecting the antibody mixture of step (d) to purification by applying anion exchange chromatography, wherein the pH setting is at least one of steps (a) to (e). It may be carried out, preferably, at least two, at least three, at least four, or all five, and most preferably, the mixed solution of the purified antibody of step (b) at an acidic pH After the step of inactivation (Virus Inactivation, VI), may be performed before step (c), but is not limited thereto.

According to one embodiment of the present invention, as a result of performing the pH of the antibody mixture solution from each purification step, the antibody mixture solution from each purification step was left at a constant pH to compare the effect of adjusting the content of the isomeric antibody, It was confirmed that the content of the acidic isomeric antibody, which was lower than the control drug, was increased by the pH standing over the entire process, thereby having a content similar to that of the control drug (Example 3). In addition, in the case of the antibody mixture after virus inactivation, it was determined that it was advantageous in terms of control and process efficiency of isomeric antibodies than in other processes.

As another aspect, the invention provides an antibody population comprising the desired amount of isomeric antibodies prepared by the above method.

The method, antibody population and desired amount of isomeric antibodies are as described above.

In another aspect, the present invention provides a method for preparing an antibody, the method comprising the steps of (a) selecting a pH and a time at which a desired amount of isomeric antibody is prepared; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a). To provide.

Preferably, the step of selecting the pH and time of step (a) (i) raises the pH used in the stationary reaction for the same time to obtain the rate of AM increase according to the following formula at each pH, and then the AM within the desired AM% range. Selecting% as AM% after pH settling, and then selecting pH and AM rise rates to be used for settling; And

[AM Rising Rate Calculation Formula]

Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

(ii) using the AM rate of rise to determine the settling time according to the following equation:

[Political time calculation formula]

pH settling time (hr) = (target AM%-AM% before pH settling) ÷ The AM rise rate selected in step (i), wherein the method, antibody population, isomer antibody, AM rise rate, settling time, etc. As described above.

In another aspect, the present invention provides a method for preparing an antibody, the method comprising the steps of (a) selecting a pH at which a desired amount of isomeric antibody can be prepared; And (b) allowing the sample containing the mixed solution of the antibody to remain within the pH selected in step (a), to control the content of the isoform in the antibody population or to a desired content. In a method for producing an antibody population comprising isomeric antibodies, a method is provided for determining pH settling time using an hourly rate of increase of a certain main acidic peak (AM).

Preferably, the pH selection and settling time determination is performed by (i) raising the pH used for the stationary reaction for the same time to obtain an AM rising rate according to the following formula at each pH, and then adjusting the AM% within the desired AM% range to pH Selecting the percentage of AM after standing, and then selecting the pH and AM rising rate to be used for standing; And

[AM Rising Rate Calculation Formula]

Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

(ii) using the AM rate of rise to determine the settling time according to the following equation:

[Political time calculation formula]

pH Settling Time (hr) = (Target AM%-AM% Before pH Settling) ÷ AM Ascent Rate Selected in Step (i) above.

It can be by.

The method, antibody population and isomeric antibody, AM rise rate, settling time and the like are as described above.

This is because the determination of pH settling time requires calculation of pH maturation time in consideration of production process time.

According to an embodiment of the present invention, the AM growth rate (% / hr), that is, the AM increase rate, was calculated using the increase rate of the main acidic isomer (Main Acidic peak, AM) increased by pH standing. Even if the content of specific isomer antibody in the culture or purified liquid was produced low by using the AM rise rate, a method was developed to determine the settling time by substituting the AM rise rate. The process time was simplified to complete the pH settling process. Using the above results, the antibody production process including the actual pH settling was carried out on the 1000 liter production scale, and when the trastuzumab manufacturing process including the pH settling was carried out through the actual applied process, the content of the isomeric antibody was sufficiently controlled on the production scale. It was confirmed that it was possible (Examples 4 and 5).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Confirmation of Isomer Antibody Control by Changing pH

Example 1.1 pH Effect Experiment for Modulating Isomer Antibodies

In the case of Trastuzumab process sample, when the acid isomer antibody content is lower than that of the reference drug, the acid isomer antibody is adjusted to have a content similar to that of the reference drug. Was performed.

Specifically, in order to confirm whether the acid isomer antibody content of the antibody mixture increases as the pH is increased, the change in the isoform of the antibody is confirmed in the antibody mixture from pH 6.0 to sequentially 8.0. In this antibody mixture, a sample having a lower acidic isoform (charge isoform) of 2% compared to the reference drug was used. As shown in Table 1 below, to raise the pH to more than 7.0, using 25mM TrisHCl buffer was diluted with buffer to pH 7.0, 7.2, 7.5, 7.7, 8.0. The initial antibody mixture was 25mg / ml, and diluted 5 times with dilution buffer for each pH to 5mg / ml. When the target pH was not measured at the time of dilution, the target pH was raised to 2M Tris, and pH 6.0 diluted with 5 mM histidine buffer pH 6.0 was compared as a control. After raising the pH to the target value, the temperature was allowed to stand at 25 ° C., and 0.5 ml of 0hr, 4hr, 8hr, and 24hr, respectively, were stored at −20 ° C. or lower until just before analysis. Changes of the isomeric antibodies, including acidic isomers, were confirmed using CEX HPLC (Cation Exchange High Performance Liquid Chromatography) analysis, and the conditions are shown in Table 2 below.

Table 1 Increase confirming acid isomer antibody (pH 7.0 ~ 8.0 condition)

pH influence experiment	pH	Buffer	Vol (ml)	Conc. (Mg / mL)	Temp ℃	Remarks
PC	pH 6.0	5 mM HistidineHCl pH 6.0	5	5	25	Sampling Time0 hr4 hr8 hr24 hr
P-1	pH 7.0	25 mM TrisHCl pH 7.0	5	5	25
P-2	pH 7.2	25 mM TrisHCl pH 7.2	5	5	25
P-3	pH 7.5	25 mM TrisHCl pH 7.5	5	5	25
P-4	pH 7.7	25 mM TrisHCl pH 7.7	5	5	25
P-5	pH 8.0	25 mM Tris-HCl pH 8.0	5	5	25

TABLE 2 CEX HPLC column conditions

	CEX HPLC
Column	ProPac TM WCX-10 column (4 * 250nm, Dionex)
HPLC	Agilent	1200 series (Agilent)
Detector	DAD (214 nm)
Mobile phase	A: Monobasic Na-phosphate, Dihydrate 0.77g / ℓ Dibasic Na-phosphate, Dihydrate 2.66g / ℓB: Buffer A + NaCl 5.84g / ℓ
Injection Vol.	50 μl
Flow-rate	0.8 ml / min

Columns for CEX-HPLC (Cation Exchange High Performance Liquid Chromatography) analysis were performed using a ProPac ™ WCX-10 column (Dionex). After dilution to 1mg / ㎖ using 10mM sodium phosphate buffer, pH 6.0 as a sample dilution buffer and 50μL injection was analyzed (Table 2). The buffer used for the analysis was 10 mM sodium phosphate, pH 7.5 (buffer A) and 10 mM sodium phosphate, 100 mM sodium chloride, pH 7.5 (buffer B), and analyzed using the buffer A and buffer B under the conditions shown in Table 3 below. . The analysis wavelength is 214 nm and HPLC instrument was used Agilent 1200 series (Agilent).

TABLE 3 CEX HPLC Analysis Conditions

Minutes	Buffer A (%)	Buffer B (%)	Flow rate (ml / min)	Program
0	85	15	0.8	Initial condition and sample injection
30	45	55	0.8	Linear gradient to 55% B
35	45	55	0.8	Hold at 55% for 5minutes
36	0	100	0.8	Step to 100% B
44	0	100	0.8	Hole at 100% B
45	85	15	0.8	Return to initial condition
55	85	15	0.8	Re-equilibrate for 10 minutes

As a result, it was confirmed that the higher the pH, and the longer the reaction time, the higher the content of the acidic portion of the acidic isomeric antibody. In particular, it was confirmed that AM (main acidic peak) in the acidic portion was raised with a certain rule depending on pH and time. In other words, as the pH is increased, the increase in AM (main acidic peak) is also increased. The peaks other than AM have little or no increase, so that the homogeneity with the reference can be increased by adjusting the pH. It was confirmed (FIGS. 1A and 1B).

Example 1.2 pH Optimization Analysis Through pH Maturation Reaction for Isomer Antibody Control

The present inventors confirmed that, through Example 1.1, the acidic peak was increased by pH maturation from pH 6.0 to pH 8.0 using the trastuzumab process solution, thereby increasing the equivalence with the reference drug.

Thus, an experiment was performed to confirm whether QC (quality control) was improved by pH maturation at pH 7.8 or higher. Specifically, process samples were raised to pH 7.8, pH 8.0, pH 8.2, pH 8.4, and pH 8.6 using 2M Tris, and 20 ml of each sample was allowed to stand at a temperature of 22-25 ° C. for 24 hours. Sampling was carried out once every 12 hours, 24 hours, and confirmed the change of isomer antibody by CEX HPLC. The range of acidic, main, basic portion on CEX HPLC of trastuzumab is shown in FIG. 2.

As a result, the AM (main acidic peak) content of the initial 0 hour sample and the total content of the acidic portion were 7.59% and 16.08%, respectively, and the AM and acidic portion contents of the reference drug were 12.45% and 20.25%, respectively. Was about 5% and 4% lower than the reference drug, respectively. As a result of analyzing the sample subjected to pH maturation for 12 hours and 24 hours by adjusting the pH from 7.8 to 8.6, it was confirmed that the AM% which was lower than the reference drug increased over all the pH ranges, and peaks other than the AM peak similar to the reference drug were increased. They could confirm that there is no big change. In particular, in case of pH 8.6, it was confirmed that AM% reached the peak of the reference drug within 12 hours, and it was confirmed that AM% reached the content of the reference drug within 24 hours even at pH 8.2 or higher (FIGS. 3A and 3B and 4A). And 4b, and Table 4).

Table 4 Changes in CEX HPLC values of pH maturation samples using clinical phase 3 process samples (% content)

Number	AM	Acidic	Main	Basic
pH 7.8 (0h) Control	7.59	16.08	79.09	4.83
pH 7.8 (12h)	9.42	18.15	76.61	5.24
pH 8.0 (12h)	9.97	18.86	76.03	5.12
pH 8.2 (12h)	10.82	23.68	71.24	5.08
pH 8.4 (12h)	11.67	21.05	73.95	5.00
pH 8.6 (12h)	12.90	22.73	72.46	4.80
Ref 0713 (Great Treaty)	12.45	20.25	72.77	6.98
pH 7.8 (24h)	10.49	19.60	75.20	5.21
pH 8.0 (24h)	11.17	20.38	74.46	5.15
pH 8.2 (24h)	12.05	21.41	73.59	5.01
pH 8.4 (24h)	13.12	22.83	72.29	4.88
pH 8.6 (24h)	15.00	25.60	69.76	4.63
Ref 0713 (Great Treaty)	12.45	20.25	72.77	6.98

# AM: main acidic peak

In addition, it was confirmed that the content of the main portion was relatively decreased as the AM% was increased, and the variation of the basic portion was less than 0.4%, and there was no significant change depending on the pH. Therefore, it can be inferred that the main portion is converted to the main acidic peak (FIGS. 5A to 5D). Through the results, it was confirmed that selecting the appropriate pH and the appropriate time according to the content pattern of the isoform of the initial sample, the quality can be matched with a similar content as the reference drug. In addition, when the time was set based on the AM (main acidic) content%, it was inferred that the maturation time and pH could be determined simply.

Example 2 Control of Content of Isomer Antibodies by pH Maturation in Culture

This experiment was carried out to determine whether the content of the isomeric antibody can be controlled by pH maturation in the culture. As such, when pH maturation is performed in the culture medium, the pH is increased in the incubator or harvest tank, and no additional equipment is required in the process equipment. It has the advantage of being sufficiently removable in the purification process.

Specifically, the experiment proceeded to the procedure shown in Table 5. The culture medium was removed using a depth filter, the pH was raised to 7.5 using 1N NaOH. After the pH was raised to 7.5, 200 ml was dispensed into a 250 ml bottle, and the temperature was pH maturated by static reaction in 25 ° C., 30 ° C. and 37 ° C. incubator, respectively. Thereafter, every 3 hours, 6 hours, 9 hours, and 24 hours, the samples were purified by rProtein A for desalting and analyzed for analysis.

Table 5 PH maturation procedure using culture

Culture	Trastuzumab Culture (8-Day Culture) Culture Volume: 5ℓ	Maturation Condition
Experiment method	1) Remove 1 cel of culture medium using Depth filter 2) Adjust culture supernatant to pH 7.5 with 1N NaOH 3) Put each bottle in 200ml and stand at 25 ℃, 30 ℃, 37 ℃ , Sampling was performed every 0hr (control), 3hr, 6hr, 9hr, 24hr.	1) Maturation pH: 7.5.2) Maturation temperature: 25 ° C, 30 ° C, 37 ° C. 3) Maturation time: 3, 6, 9, 24hrs.
Purification Steps for Analysis	4) Sampling samples were purified by Protein A with an open column for analysis. 4-1) 10 ml of the culture supernatant was loaded into 1 ml of Protein A FF. 4-2) 5CV wash with 25 mM TrisHCl pH 7.2 and 150 mM NaCl. 4-3) 5CV wash 2 with 25 mM TrisHCl pH 7.2, (w / o NaCl) 4-4) Elution with 4 ml of 0.1M glycine HCl pH 3.0 4-5) Neutralizing with 2M Tris 4- 6) Desalt to 20mM Na phosphate pH 6.0 and concentrate to 1mg / ml. 4-7) Analyze the sample by CEX HPLC.

Table 6 Changes in CEX HPLC values of pH maturation samples in culture (% content)

Number	AM	Acidic	Main	Basic
Control	9.18	19.71	72.48	7.81
25 ° C (3h)	9.46	20.18	71.41	8.41
30 ℃ (3h)	9.66	20.56	71.32	8.12
37 ° C (3h)	10.12	21.08	71.63	7.29
25 ℃ (6h)	9.73	20.87	71.21	7.92
30 ℃ (6h)	10.37	21.71	71.04	7.25
37 ℃ (6h)	11.08	23.07	69.46	7.47
25 ° C (9h)	10.00	21.44	71.42	7.15
30 ℃ (9h)	10.99	23.07	69.09	7.84
37 ° C (9h)	11.90	25.92	66.26	7.81
25 ℃ (24h)	11.10	22.59	69.23	8.18
30 ℃ (24h)	12.87	25.97	66.07	7.96
37 ° C (24h)	14.72	32.22	60.26	7.52
Ref. H0713 (great treaty)	12.45	21.05	72.40	6.55

As a result, as shown in the analysis results of Table 6, it was confirmed that the AM (main acidic peak) increased with the maturation temperature. In addition, it was confirmed that AM% was constantly increased over time (Table 6, FIGS. 6A and 6B, and FIGS. 7A and 7B). However, the disadvantage is that not only the AM (main acidic peak) rises above 30 ° C., but also the base line of the acidic portion also rises (FIGS. 7A and 7B). Particularly, at 25 ° C., the main acidic peak was selectively increased compared to other acidic peaks, but from 30 ° C. and above, the entire acidic portion as well as the main acidic peak was increased (FIGS. 6A and 6B, and FIGS. 7A and 7B). From these results, it was confirmed that the selectivity was lowered at 30 ° C. or higher in the selective increase of the lower main acidic peak than the reference drug.

Example 3 Control of Content of Isomer Antibodies by pH Maturation in Purified Liquid

It was confirmed through the Examples 1 and 2 that the acidic isomer antibody (acidic form) increases when the pH is increased by maturation. This was followed by a control experiment of isomeric antibodies by pH maturation in the purified solution. In particular, pH maturation experiments were carried out for each step of purification, and evaluation of which step was most advantageous was conducted. Figure 8 shows a step candidate group that can be applied by inserting the pH maturation in the existing Trastuzumab purification process of the present inventors.

Specifically, pH maturation is applied after each of CEX (Cation Exchange Chromatography), VI (Virus Inactivation), HIC (Hydrophobic Interaction Chromatography), UF / DF (Ultrafiltation and Diafiltration), and AEX (Anion Exchange Chromatography). Each step was compared with the charge isoform control effect of isomer antibody by pH maturation.

Example 3.1 Analysis of pH Maturation Effect in CEX (Cation Exchange Chromatography) and VI (Virus Inactivation) Steps

PH maturation was applied to the purified liquid from the CEX column and VI (virus inactivation) process. The temperature was carried out at 4 ℃, 25 ℃ the temperature encountered in the process (Tables 7 and 8).

TABLE 7 PH maturation procedure in CEX and VI steps

Purification steps	order	Maturation Condition
CEX (Cation Exchange chromatography) Step	1) 1 liter of culture was removed using a Depth filter. 2) The culture supernatant from which the cells were removed was lowered to pH 5.0 using 10% acetic acid and held for 1hr. 3) Using the same Depth filter, precipitation was performed. 4) The conductivity was adjusted to 6mS / cm for loading on the CEX column, and then sterilized and filtered. 5) pH was adjusted to 2M Tris using elute purified using CEX column (Fractogel COO (M)). Raised to 8.0, pH maturation test was performed.	Maturation temperature: 4 ° C, 25 ° C. Maturation pH: 8.0. Maturation time: 6, 11, 24hrs.
VI (Virus Inactivation) Step	1) Using the CEX elute prepared in the above step was reduced to 1M citric acid to pH 3.8 and then held for 1 hr. 2) After completion of holding, the pH was adjusted to pH 8.0 using 2M TrisHCl and pH maturation was performed for each pH maturation time.	Maturation temperature: 4 ° C, 25 ° C. Maturation pH: 8.0. Maturation time: 6, 11, 24hrs.

            

Table 8 PH maturation condition at CEX and VI step

Cation Exchange Chromatography				Virus Inactivation
No.	Temp ℃	Time (hr)	Volume	No.	Temp ℃	Time (hr)	Volume
CEX-control		0	30	VI-Control		0	30
C-1	4 ℃	6	30	V-1	4 ℃	6	30
C-2	4 ℃	11	30	V-2	4 ℃	11	30
C-3	4 ℃	24	30	V-3	4 ℃	24	30
C-4	25 ℃	6	30	V-4	25 ℃	6	30
C-5	25 ℃	11	30	V-5	25 ℃	11	30
C-6	25 ℃	24	30	V-5	25 ℃	24	30

Table 9 Isomer antibody content% with time by pH maturation in CEX and VI step

Step	Number	AM	Acidic	Main	Basic
CEX	CEX Control	8.95	19.06	74.10	6.84
	CEX 4 ℃ (6h)	9.98	20.76	72.33	6.91
	CEX 4 ℃ (11h)	10.19	20.87	72.48	6.65
	CEX 4 ℃ (24h)	10.62	21.49	71.90	6.61
	CEX 25 ℃ (6h)	10.79	21.73	71.66	6.61
	CEX 25 ℃ (11h)	11.49	21.87	71.66	6.47
	CEX 25 ℃ (24h)	12.48	24.18	69.42	6.40
VI	VI Control	9.48	19.96	73.22	6.81
	VI 4 ℃ (6h)	9.83	19.93	73.39	6.69
	VI 4 ° C (11h)	10.10	20.19	73.15	6.66
	VI 4 ℃ (24h)	10.71	21.46	71.99	6.55
	VI 25 ℃ (6h)	10.80	21.35	72.13	6.52
	VI 25 ℃ (11h)	11.61	22.71	70.84	6.45
	VI 25 ℃ (24h)	12.78	24.52	69.10	6.38
Ref	Ref.H0713	12.66	21.50	71.82	6.69

As a result, pH maturation using the sample after the CEX process as shown in Table 9, Figures 9a to 9d, and Figure 10, AM% is 8.95% to 25 ℃, 12.48% after 24 hours 12.66% of the reference drug Almost similar level with.

In the case of process samples after VI, when pH maturation was performed at 25 ° C. and after 24 hours, the AM% increased from 9.48% to 12.78%, similar to the AM% of the reference drug. It was confirmed that AM increased with time compared to the initial 0 hour control in both CEX and VI steps at 4 ℃. After 24 hours, it increased to 10.62% and 10.71%, respectively, but it was relatively lower than 25 ℃. Ascending rate was shown, and AM% was similar in 6 hours condition at 25 ° C and 24 hours condition at 4 ° C. In the case of the main portion, the content was decreased in both CEX and VI conditions at 25 ° C over time, and after 24 hours, it was reduced by 4% compared to the initial control, and there was almost no difference between the two steps. In the case of the basic portion, no appreciable increase or decrease was observed.

Therefore, the effect was similar when the pH maturation was tried in both processes. Particularly, in the VI step, there is an existing process of naturally increasing the pH for neutralization after the pH down, so when the pH is adjusted to 8.0 in the VI process, pH maturation can be performed without adding a process.

From the above results, it was confirmed that it is advantageous to apply pH maturation in the VI step in terms of simplification of the process.

Example 3.2: Analysis of pH maturation effect in HIC (Hydrophobic Interaction Chromatography)

PH maturation was performed in the procedure of Table 10 in the HIC purification step of the purification process.

Table 10 PH maturation procedure in HIC step

Purification stage	order	Maturation Condition
Sample information	1) Trastuzumab culture solution (7day) Culture Volume: 5ℓ2) Sample purified using Fractogel COO (M) column	Other conditions
Hydrophobic Interaction (HIC) Step	1) Fractogel COO (M) elute was mixed with the same amount of 60mM Na Acetate + 1.2M Nacitrate pH 6.0, and 2) loaded with Phenyl sepharose FF. 4) The HIC elute was raised to pH 8.0 with 1M NaOH. 5) Maturation temperature was carried out at 4 ° C and 25 ° C. Sampling was performed for 6 ~ 24 hours to CEX the pH maturation pattern. Analyzed by HPLC.	Maturation temperature: 4 ° C, 25 ° C. Maturation pH: 8.0. Maturation time: 6,9,24hrs.

Table 11 Isomer antibody content% with time by pH maturation in HIC step

Step	Number	AM	Acidic	Main	Basic
HIC	HIC Control	8.51	20.46	71.94	7.60
	HIC 4 ℃ (6h)	9.53	21.16	72.17	6.67
	HIC 4 ℃ (9h)	9.85	21.75	71.86	6.39
	HIC 4 ℃ (24h)	10.60	22.48	71.31	6.21
	HIC 25 ℃ (6h)	10.09	20.89	72.54	6.57
	HIC 25 ℃ (9h)	10.71	22.56	71.07	6.37
	HIC 25 ℃ (24h)	11.58	23.88	69.90	6.23
Ref	Ref.H0713	12.30	20.28	72.36	7.37

As a result, as shown in Table 11, FIGS. 11A to 11D, and FIG. 12, when pH maturation was performed in HIC elute, it was confirmed that AM (Main acidic peak) increased in time. Compared to the control 8.51%, it increased to 10.09% at 25 ° C. after 6 hours and 11.58% at 25 ° C. after 24 hours. However, in case of 4 ℃, the rate of increase of AM was significantly lower than that of 25 ℃, and after 24 hours, the rate of increase was 10.60%, which was similar to that of 6 hours of 25 ℃, indicating a relatively low rate of rise.

In the case of the main portion, the content was reduced at 25 ℃, after 24 hours was confirmed to decrease by about 2% compared to the control. The basic portion was reduced by about 1% compared to the control, and no difference was observed in all conditions. However, the pH maturation in HIC resulted in lower effect than CEX or VI. In the CEX or VI step, the content was similar to that of the reference drug after 24 hours based on AM (main acidic peak), but the pH maturation of HIC samples was 1% less than that of the reference drug. In particular, when looking at the CEX HPLC pattern of the sample after 24 hours, it can be seen that the acidic base line is elevated (Fig. 12). This is because HIC elute contains high salt and 2M Tris is not able to raise pH to 8.0 due to weak buffering when raising the pH, and it may have a negative effect on the product because the pH should be increased by adding 1N NaOH. Because.

Therefore, pH maturation in HIC step is more efficient in pH maturation with longer time and higher temperature, but it has disadvantage of high pH with strong base because it contains high salt. It has been confirmed that there may be disadvantages in terms of aspects.

Example 3.3: Analysis of pH maturation effects in UF / DF1

The experiment for the case where pH maturation is performed in UF / DF1 was performed by the procedure of Table 12.

Table 12 Isomer antibody content% with time by pH maturation in HIC step

Sample information	1) Culture (7day) Culture Volume: 5ℓ 2) Sample purified using CEX column 3) Sample purified using HIC column	Maturation Condition
pH maturation step at UF / DF1 (Ultrafiltration & Diafiltration I)	1) Proceed with UF / DF1 with 25mM TrisHCl 7.5. 2) Adjust pH 8.0 with 2M Tris and proceed with pH maturation.	Maturation temperature: 4 ℃, 25 ℃. Maturation time: 6,24hrs.

Table 13 Isomer antibody content% with time by pH maturation in UF / DF1 step

Step	Number	AM	Acidic	Main	Basic
UF / DF1	UF / DF1 Control	9.22	21.05	72.77	6.18
	UF / DF1 4 ℃ (6h)	9.67	20.72	73.10	6.19
	UF / DF1 4 ℃ (24h)	10.11	21.30	72.72	5.98
	UF / DF1 25 ℃ (6h)	10.45	21.64	72.33	6.03
	UF / DF1 25 ℃ (24h)	11.71	23.37	70.63	6.00
Ref	Ref.H0713	12.43	21.11	71.91	6.98

As a result, as shown in Table 13, Figure 13a to 13d, and Figure 14, when the pH maturation in UF / DF1, it was confirmed that in the case of AM (main acidic peak) rises in time. Compared to the control 9.22%, it increased to 10.45% at 25 ° C after 6 hours and 11.71% at 25 ° C after 24 hours, approaching 12.43% of the control drug. In case of 4 ° C, the rate of AM rise was significantly lower than that of 25 ° C. After 24 hours, the rate of AM rise was 10.11%, which was increased to a content similar to that of 6 hours at 25 ° C. In the case of the main portion at 25 ℃, after 24 hours it was confirmed that the reduction by 2% compared to the control. There was no significant difference in the basic portion compared to the control.

In other words, when pH maturation was performed in UF / DF1, AM showed a 1% lower increase compared to CEX or VI for 24 hours. Elevation of the acidic and basic base lines showed much less elevation than in HIC. Therefore, it may be advantageous to perform pH maturation at this step rather than HIC. However, since the step of contacting the high pH at the previous stage of the final column is limited to the steps that can be removed when the fragment or aggregation occurs in this condition, there may be a disadvantage that the risk must be carried out as much.

Example 3.4 Final Evaluation of pH Maturation Steps in the Purification Process

Based on the experimental results in the purification step, we compared which step is the most favorable step to apply pH maturation by application score. The evaluation items were evaluated by scoring QC (quality control) of the isomeric antibody, process adaptation to the existing process, risk of aggregation during application, and ease of stopping the pH maturation reaction.

As a result, HIC is slightly disadvantageous in terms of charge isoform quality control and similar quality control is performed in the remaining steps. In HIC, not only AM (main acidic peak) but also acidic and basic base line were observed to rise together. In terms of process adaptation, HIC gave a low score due to the problem of excessive 1M NaOH in raising the pH, and CEX gave a slightly lower score due to the process unnecessary to increase the pH and lower it again for VI. In terms of aggregation, HIC contains a lot of salt, NaOH also contains a lot of high risk of aggregation occurred that gave a low score. UF / DF1 and AEX were penalized as they are after the HIC process where most of the aggregation can be removed. In pH down for quenching after pH maturation, CEX has a conventional pH down process in VI process, and after pH maturation in VI, 2X buffer (pH 6.0) is mixed for HIC Load prep. In order to lower the progression without further processing gave a high score. The final score was therefore assessed as VI> CEX> AEX> UF / DF1 >> HIC (Table 14).

Table 14 Evaluation table for each purification step pH maturation

Process	Quality Control	Process Adaptation	Aggregation Risk (the lower the score, the higher the score)	pHQuenching	Total
CEX	*****	***	****	****	16
VI	*****	****	****	****	18
HIC	***	***	**	***	11
UF / DF1	*****	****	***	**	14
AEX	*****	****	***	***	15

Example 4 Establishment of Methods for Determining In-Process pH Maturation Times

Experiments were performed to determine the pH maturation time in the VI step, which was evaluated as advantageous with pH maturation. First, the reference drug was analyzed to calculate the time of pH maturation. A total of five controls were analyzed by CEX HPLC.

Table 15 Isomer Antibody Content Analysis of the Reference Drug

Reference Drug		Area content (%) at CEX HPLC
		Acidic portion			Main portion		Basic portion
Label	Reference Lot	AM	A2	Acidic sum	M1	Main sum	B1	B2	Basic sum
R1	H0666B01	11.96	3.67	21.71	7.69	71.70	4.89	1.70	6.59
R2	H0750B01	12.81	3.70	22.63	8.01	70.48	4.82	1.57	6.90
R3	H0717B01	12.91	3.71	22.56	7.87	70.93	4.89	1.32	6.51
R4	B1600B01	12.14	3.82	21.49	8.05	72.51	4.70	1.23	6.00
R5	H0713B01	12.46	3.31	20.68	7.04	72.86	4.83	1.37	6.46
STDV		0.41	0.20	0.81	0.41	1.01	0.08	0.19	0.32
Average		12.45	3.64	21.81	7.73	71.70	4.83	1.44	6.49

            

As a result, the percentage of main acidic peak (AM) was 12.45% on average, 21.8% for acidic portion, 71.7% for main portion, and 6.49% for basic portion (Table 15). In particular, since the main acidic peak (AM) rises depending on the pH and time as a result of the above embodiments, it can be used as a time determination criterion for pH maturation using AM%.

Example 4.1: Determination of pH maturation factor (AM growth rate)

In order to calculate the pH maturation time in the VI step using the AM (main acidic peak) content, AM growth rate was calculated, which means the amount (%) of the AM rising time. To obtain this value, the cultures (7 days) were collected, and the samples purified on CEX column (Fractogel COO (M)) were lowered to pH 3.8 for VI, and then gradually increased to pH 7.8 to 8.6 after 1hr inactivation, respectively. Samples were collected in ml. Samples sampled at 30 ml for each pH were maturated at room temperature (18 ~ 23 ℃) for 24 hours, and the AM growth rate (% / hr) was divided by the reaction time (24 hours) of the AM difference after maturation and before maturation. ) Was calculated (Table 16).

Table 16 Calculation of AM growth rate for pH maturation in VI step

Label (pH)	AM (%) before pH maturation	AM (%) after pH maturation	Maturation Time (hr)	AMgrowth rate (% / hr)
pH 7.8	7.587	10.487	24	0.12
pH 8.0	7.587	11.17	24	0.15
pH 8.1	7.587	11.68	24	0.17
pH 8.2	7.587	12.05	24	0.19
pH 8.4	7.587	13.116	24	0.23
pH 8.6	7.587	15.003	24	0.31
Reference	12.45	N / A	N / A	N / A

As a result, as shown in Table 16, as the pH was increased, the AM growth rate was increased. Especially, when the pH was within 11% or more, within 12.45% of the AM, the proper pH was 8.0 ~ The scope of 8.2 was deemed appropriate. AM growth rate of pH 8.0 ~ 8.2 was 0.15 ~ 0.19% / hr, and 0.17% / hr was calculated at median pH 8.1.

In order to evaluate experimentally whether the AM growth rate is practically appropriate in the pH range of 8.0 to 8.2, a verification experiment was conducted for 2 batches on a small scale. The initial% of AM was 6.81% and 6.65%, respectively, and pH maturation was performed at room temperature at pH 8.0, sampling every 3, 6, 9, 13, and 24 hours, and CEX HPLC analysis was carried out as well as acidic form, The main and basic forms were monitored (Table 17). The time was plotted based on the AM% averaged over 2 batches. As a result of plotting and calculating the regression curve for the spot, it was confirmed that the slope was in the range of 0.15 to 0.17, which is the AM growth factor of pH 8.0 to 8.1 calculated above (Fig. 16).

Table 17 Change of Isomer Antibody Content (%) by pH maturation Application in VI Step in 2 Batch

Label	Time	AM	A2	Acidic sum	M1	Main sum	B1	B2	Basic sum
Batch
	1	0	6.81	3.89	15.27	6.65	78.92	3.99	1.62	5.81
		3	7.42	3.98	16.21	6.73	76.93	4.26	2.23	6.86
		6	8.08	3.97	16.76	6.70	77.30	4.22	1.72	5.94
		9	8.52	3.93	17.11	6.72	77.14	4.19	1.56	5.75
		13	9.13	3.99	17.98	6.80	75.98	4.29	1.74	6.04
24		10.72	3.95	18.91	6.78	75.37	4.25	1.48	5.73
Batch 2	0	6.65	3.57	12.28	7.32	81.68	4.56	1.93	6.04
	3	7.37	3.59	12.81	7.37	80.62	4.67	1.90	6.57
	6	8.18	3.69	14.11	7.37	79.29	4.74	1.86	6.60
	9	8.84	3.73	15.00	7.53	77.93	4.87	2.20	7.07
	13	9.33	3.71	15.52	7.48	77.98	4.73	1.77	6.50
	24	10.92	3.81	17.41	7.67	75.65	4.84	2.10	6.94
Average (Batch1 & Batch2)	0	6.73	3.73	13.78	6.98	80.30	4.27	1.77	5.92
	3	7.39	3.79	14.51	7.05	78.78	4.46	2.06	6.72
	6	8.13	3.83	15.44	7.04	78.29	4.48	1.79	6.27
	9	8.68	3.83	16.05	7.12	77.54	4.53	1.88	6.41
	13	9.23	3.85	16.75	7.14	76.98	4.51	1.76	6.27
	24	10.82	3.88	18.16	7.23	75.51	4.55	1.79	6.34

Therefore, using this AM growth rate, it was confirmed that pH and maturation time for controlling the content of isomer antibody from the content of isomer antibody before pH maturation can be determined.

Example 5: pH maturation verification at production scale

In order to check whether the pH maturation method for controlling the content of the isomer antibody confirmed in the above examples is applicable in the purification process of the production scale to which trastuzumab is applied, the present pH maturation method was introduced on a 1000 L culture scale.

Purification processes for the preparation of Trastuzumab include Cation Exchange Chromatography (CEX), Virus Inactivation (VI) by low pH, Hydrodrophobic Interaction Chromatography (HIC), and Ultrafiltration. Consists of 1st Ultrafiltation and Diafiltration (UF / DF I), Anion Exchange Chromatography, 2nd Ultrafiltration and Diafiltration II (UF / DF II), VF (Virus Filtration), Final Formulation It is.

Among these processes, pH maturation was performed after VI (Virus Inactivation), which was determined to be most advantageous during pH maturation. A brief summary of the process is shown in Table 18 below.

Table 18 Process Progress on 1000 L Production of Trastuzumab (HD201) with pH maturation

Process		Specification
Batch number		HD201-TRS13001
Culture Size		1000ℓ
Clarification	Depth
	1	Cell removal
Depth2	pH down (pH 5) → Depth filteration
CEX	Column	Fractogel COO (M)
		Pall Φ700
		Vol: 58L (14.5cm)
	Protein load (mg / ml)	28
	Flow rate (cm / hr)	150
	CEX Pool	A2 + A3 + main
VI	Low pH	3.8 (pH down)
pH maturation	High pH	8.1 (18 ~ 24 ℃)
HIC	Column	Phenyl Sepharose FF
		Pall Φ700
		Height: 13cm
	Protein load	29
	Flow rate (cm / hr)	150
UF / DF1	Membrane	Pellicone	3 * 8 EA
	Dilution Factor
	2 ^ 5
AEX	Column	Q FF
		Vol: 14ℓ
		BPG Φ300
	Protein load	115
	Flow rate (cm / hr)	150
pH maturation		pH 8.0 (24hr)
UF / DF2	Membrane	Pellicone	3 * 8 EA

Example 5.1: Calculation of pH maturation time on production scale

The maturation time was calculated and maturation proceeded as follows for pH maturation during VI process at 1000ℓ production scale. The initial AM% criterion was based on the CEX elute sample, and the target AM% was applied to 10.5%.

(1) Content of AM (main acidic form) of CEX elute%: 7.11%

(2) Target AM (main acid form) content%: 10.5%

The reason why the target AM (main acid form) content% is 10.5% is because the UF / DF1 and AEX processes operate at pH 7.5, which results in an average AM increase of more than 1% due to the high pH. Therefore, we gave a safety margin to reflect the rise of the process after pH maturation, so the target in VI was determined to be 10.5%.

(3) AM growth factor: This factor calculates the rate of increase of AM (main acidic peak), which is a target peak for pH maturation, per hour. This growth factor was determined through data analysis through the scale down experiment, and the conditions were designed to complete pH maturation within 24hr. This factor is a factor dependent on the maturation pH. At pH 8.1, 0.17% / hr meets Target AM% (10.5%) in 24hr.

1000ℓ scale batch

# pH maturation process time (hr) = (Target AM%-AM% before VI) ÷ 0.17 (Factor:% / hr)

= (10.5-7.11) ÷ 0.17 (Factor:% / hr)

= 20hr (± 2hr)

# Margin ± 2hr is the range set by 2 times considering the time that it takes more than 1hr to add 2X HIC load prep buffer to stop the pH maturation reaction.

As a result of pH maturation in VI step under the above conditions, the AM% after UF / DF2 including pH maturation was 11.53% at 7.11%, which is AM% immediately before pH maturation. It was confirmed that it was maintained at ± 1.3 at 12.5%, AM% of the reference drug (Table 19 and FIGS. 17A and 17B). Termination of pH maturation was terminated by mixing buffer (1.2M Na citrate + 60mM acetate pH 6.0) in a 1: 1 ratio, and the termination pH was 6.2.

Table 19 PH maturation conditions and results at 1000 l production scale

Batch number		HD201-TRS13001
USP Scale		1000ℓ
DSP Process Step		After VI
pH Maturation Condition	Aging Time (hr)	20
	Temp (℃)	21-25 (23)
	Maturation pH	8.1
	Target AM (%)	10.5
	AM growth factor (% / hr)	0.17
Before pH maturation (VI)	AM (%)	7.11
	Acidic (%)	15.24
	Main (%)	78.81
	Basic (%)	5.95
After pH maturatiom (UF / DFⅡ)	AM (%)	11.53
	Acidic (%)	20.61
	Main (%)	73.22
	Basic (%)	6.16

            

Therefore, the above results indicate that the pH maturation method of the present invention is sufficiently applicable to the production scale, and that the present method not only can produce high-quality antibodies, but also has the same quality as that of the reference drug in terms of biosimilar production. It supports the production of antibodies.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (21)

1. (a) selecting a pH and time to prepare a desired amount of isomeric antibody; And

   (b) allowing a sample containing a mixture of antibodies to remain within the pH selected in step (a) for a selected time period, the desired population of antibody populations comprising a charge isoform. Manufacturing method.
2. The method of claim 1, wherein the mixed solution of the antibody is a culture solution or a purified solution for producing the antibody.
3. The method of claim 2, wherein the culture solution is a culture solution containing cells or a culture supernatant from which cells have been removed.
4. The method of claim 1, wherein the desired amount of isomeric antibody is within a range of ± 15% of the% isomeric antibody content in the reference drug.
5. The method of claim 1, wherein the main acidic peak (AM) is in the range of ± 20% with AM in the reference drug.
6. The method of claim 1, wherein the pH is at least pH 7.0 and at most pH 9.0.
7. The method of claim 1, wherein the settling within the selected pH is performed at a temperature of 4 ° C. or more and 40 ° C. or less.
8. The method of claim 7, wherein the settling within the selected pH is performed at a temperature of at least 15 ° C. and at most 30 ° C. 9.
9. The method according to claim 1, wherein the pH settling time is 1 hour or more and 48 hours or less.
10. The method of claim 1, wherein the step of selecting pH and time of step (a)

    (i) After raising the pH used for the stationary reaction for the same time to obtain the rate of AM rise according to the following formula at each pH, AM% within the desired AM% range was selected as AM% after the pH settling, and then the pH to be used for standing Selecting a rate of increase of AM; And

    [AM Rising Rate Calculation Formula]

    Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

    (ii) using the AM rate of rise to determine the settling time according to the following equation:

    [Political time calculation formula]

    pH Settling Time (hr) = (Target AM%-AM% Before pH Settling) ÷ AM Ascent Rate Selected in Step (i) above.
11. The method of claim 1, wherein the antibody is Trastuzumab.
12. The method of claim 1, wherein the isomeric antibody is an acidic isomeric antibody.
13. The method of claim 1, wherein the pH settling step is performed in one or more of the following steps:

    (i) the mixture of antibodies is purified using Cation Exchange Chromatography (CEX);

    (ii) the mixed solution of the antibody was purified by the Virus Inactivation (VI) step;

    (iii) the mixture of antibodies was purified using Hydrophobic Interaction Chromatography (HIC);

    (iv) the mixed solution of the antibody was purified by ultrafiltration (Ultrafiltation and Diafiltration, UF / DF); or

    (v) The liquid mixture of the antibody was purified using Anion Exchange Chromatography.
14. The method of claim 12, wherein the pH standing step is performed after the mixed solution of the antibody is purified by a Virus Inactivation (VI) step.
15. The method of claim 1, wherein the method of preparing an antibody population comprising the desired amount of isoforms of the antibody comprises the following steps:

    (a) purifying a sample containing a mixture of antibodies by applying Cation Exchange Chromatography (CEX);

    (b) virus inactivation (VI) of the mixed solution of the antibody purified in step (a) to an acidic pH;

    (c) purifying the mixed solution of the virus-inactivated antibody of step (b) by applying hydrophobic reaction chromatography (HIC);

    (d) purifying the antibody mixture purified in step (c) by primary ultrafiltration (1st Ultrafiltation and Diafiltration, UF / DF I); And

    (e) subjecting the antibody mixture of step (d) to purification by applying anion exchange chromatography (Anion Exchange Chromatography), wherein pH standing is performed in at least one of steps (a) to (e) How to be.
16. The method of claim 15, wherein the pH settling is performed after step (b) but before step (c).
17. An antibody population comprising a desired amount of isomeric antibodies prepared by the method of any one of claims 1 to 16.
18. (a) selecting a pH and time to prepare a desired amount of isomeric antibody; And

    (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a), to control the content of isoforms in the antibody population.
19. 19. The method of claim 18, wherein the step of selecting pH and time of step (a)

    (i) After raising the pH used for the stationary reaction for the same time to obtain the rate of AM rise according to the following formula at each pH, AM% within the desired AM% range was selected as AM% after the pH settling, and then the pH to be used for standing Selecting a rate of increase of AM; And

    [AM Rising Rate Calculation Formula]

    Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

    (ii) using the AM rate of rise to determine the settling time according to the following equation:

    [Political time calculation formula]

    pH Settling Time (hr) = (Target AM%-AM% Before pH Settling) ÷ AM Ascent Rate Selected in Step (i) above.
20. (a) selecting a pH at which a desired amount of isomeric antibody can be prepared; And

    (b) a method of controlling the content of isoforms in a population of antibodies or isomers of a desired content comprising the step of allowing a sample containing a mixture of antibodies to remain within the pH selected in step (a). A method for producing an antibody population comprising a vaginal antibody, wherein the pH settling time is determined using an hourly rate of increase of a certain main acidic peak (AM).
21. The method of claim 20, wherein the pH selection and determination of the settling time,

    (i) After raising the pH used for the stationary reaction for the same time to obtain the rate of AM rise according to the following formula at each pH, AM% within the desired AM% range was selected as AM% after the pH settling, and then the pH to be used for standing Selecting a rate of increase of AM; And

    [AM Rising Rate Calculation Formula]

    Rate of AM rise (% / hr) = (AM% after pH settling-AM% before pH settling) ÷ Settling time (hr)

    (ii) using the AM rate of rise to determine the settling time according to the following equation:

    [Political time calculation formula]

    pH Settling Time (hr) = (Target AM%-AM% Before pH Settling) ÷ AM Ascent Rate Selected in Step (i) above.

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