CN119233988A - Anti-human growth hormone single domain antibody and application thereof - Google Patents
Anti-human growth hormone single domain antibody and application thereof Download PDFInfo
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Abstract
本发明涉及特异性结合人生长激素的单域抗体或其抗原结合片段，含有所述单域抗体或其抗原结合片段的缀合物，编码所述单域抗体或其抗原结合片段的核酸分子及包含其的宿主细胞，以及相关用途。此外，此发明涉及所述缀合物的检测或人生长激素的纯化用途。
The present invention relates to a single domain antibody or an antigen-binding fragment thereof that specifically binds to human growth hormone, a conjugate containing the single domain antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the single domain antibody or the antigen-binding fragment thereof, a host cell containing the same, and related uses. In addition, the invention relates to the use of the conjugate for the detection or purification of human growth hormone.
Description
The present invention relates to single domain antibodies or antigen binding fragments thereof that specifically bind to human growth hormone, conjugates containing the single domain antibodies or antigen binding fragments thereof, nucleic acid molecules encoding the single domain antibodies or antigen binding fragments thereof, and host cells comprising the same, and related uses. Furthermore, the invention relates to the detection of said conjugates or to the purification use of human growth hormone.
Human growth hormone (hGH) is a protein secreted by the pituitary gland cells and is a peptide hormone. Is a single peptide protein hormone secreted by human anterior She Shi acid cells, and is a peptide hormone composed of 191 amino acids. Growth hormone has been used clinically in 1958 as a specific drug for treating dwarfism, and its clinical indications are continuously expanding after large-scale production in pharmaceutical factories in the 80 th 20 th century. The Chinese medicinal preparation is widely applied to the aspects of promoting the growth of human bodies, resisting tissue and organ failure, improving the nutrition of severe patients, treating cachexia (nutrition failure), resisting infection and inflammation, promoting wound and burn healing, improving the immunity of organisms and the like.
Nanobodies are a special class of antibodies derived from camelids. In 1993, hamers-Casterman et al showed that there was an antibody in camel origin that naturally lacks the light chain and only contains the heavy chain, called the heavy chain antibody. By cloning the variable region genes of heavy chain antibodies, single domain antibodies consisting of only one heavy chain variable region, referred to as VHH antibodies, can be obtained. The VHH antibody has a crystal structure with a diameter of only 2.5nm and a length of 4nm, and is also called nanobody. Nanobodies are only one tenth of the size of conventional IgG-type antibodies, being the smallest fragment that naturally occurs and that can bind to antigen. In addition, the nano antibody has the characteristics of acid and alkali resistance, high temperature resistance, easiness in production and the like, and can be advantageously applied to immunoaffinity chromatography purification of human GH to replace the traditional human GH purification method with complicated process.
Disclosure of Invention
Through a great deal of research, the inventor of the application screens and obtains a series of anti-human growth hormone single domain antibodies, and the single domain antibodies have high binding activity with human growth hormone. In particular, the single domain antibody of the application has excellent high temperature resistance, acid and alkali resistance and excellent stability, can effectively combine with hGH under neutral condition, and can effectively dissociate with hGH under low pH acid condition, thus being advantageously applied to affinity purification of human growth hormone. In addition, the single domain antibody has the characteristics of small molecular weight, easy production and the like.
Based on this, the application also provides conjugates comprising the single domain antibodies or antigen binding fragments thereof, nucleic acid molecules encoding the single domain antibodies or antigen binding fragments thereof and host cells comprising the same, and related uses.
Single domain antibodies and antigen binding fragments thereof
Accordingly, in one aspect, the present application provides a single domain antibody or antigen-binding fragment thereof capable of specifically binding human growth hormone (hGH), said single domain antibody or antigen-binding fragment thereof comprising:
(a) CDR1 having a structure as shown in X 1X2AX3 G (formula I, SEQ ID NO: 23);
(b) CDR2 having a structure as shown in X 4IX5X6X7GX8X9TX10 YADSVKG (formula II, SEQ ID NO: 24);
(c) CDR3 having a structure as shown in AFSVTIPTRARHWVD (SEQ ID NO: 19) or ATX 11YYSDYDVPX12X13SX14EX15 DY (formula III, SEQ ID NO: 25) or SRGDX 16 GILHGVVHY (formula IV, SEQ ID NO: 26);
Wherein,
X 1 is selected from the group consisting of (i) amino acid residues a, S, N and (ii) amino acid residues that are conservative substitutions relative to (i);
X 2 is selected from the group consisting of (i) amino acid residues R, F, Y, and (ii) amino acid residues that are conservative substitutions relative to (i);
X 3 is selected from the group consisting of (i) amino acid residues M, V, and (ii) amino acid residues that are conservative substitutions relative to (i);
X 4 is selected from (i) amino acid residues a, S and (ii) amino acid residues that are conservative substitutions relative to (i);
x 5 is selected from the group consisting of (i) amino acid residues E, S, G, and (ii) amino acid residues that are conservative substitutions relative to (i);
X 6 is selected from the group consisting of (i) amino acid residues G, W, and (ii) amino acid residues that are conservative substitutions relative to (i);
X 7 is selected from the group consisting of (I) amino acid residues I, M, L, and (ii) amino acid residues that are conservative substitutions relative to (I);
x 8 is selected from (i) amino acid residues a, G and (ii) amino acid residues that are conservative substitutions relative to (i);
X 9 is selected from (i) amino acid residues T, S and (ii) amino acid residues that are conservative substitutions relative to (i);
X 10 is selected from the group consisting of (i) amino acid residues Y, V, S and (ii) amino acid residues that are conservative substitutions relative to (i);
X 11 is selected from (i) amino acid residues S, N and (ii) amino acid residues that are conservative substitutions relative to (i);
X 12 is selected from the group consisting of (i) amino acid residue V, A, and (ii) amino acid residue that is a conservative substitution with respect to (i);
X 13 is selected from the group consisting of (i) amino acid residues R, T, and (ii) amino acid residues that are conservative substitutions relative to (i);
x 14 is selected from the group consisting of (i) amino acid residues N, D, and (ii) amino acid residues that are conservative substitutions relative to (i);
x 15 is selected from the group consisting of (i) amino acid residues Y, F, and (ii) amino acid residues that are conservative substitutions relative to (i);
X 16 is selected from the group consisting of (i) amino acid residues F, Y, and (ii) amino acid residues that are conservative substitutions relative to (i).
In certain embodiments, X 1 is selected from amino acid residues A, S, N, X 2 is selected from amino acid residues R, F, Y, X 3 is selected from amino acid residues M, V, X 4 is selected from amino acid residues A, S, X 5 is selected from amino acid residues E, S, G, X 6 is selected from amino acid residues G, W, X 7 is selected from amino acid residues I, M, L, X 8 is selected from amino acid residues A, G, X 9 is selected from amino acid residues T, S, X 10 is selected from amino acid residues Y, V, S, X 11 is selected from amino acid residues S, N, X 12 is selected from amino acid residues V, A, X 13 is selected from amino acid residues R, T, X 14 is selected from amino acid residues N, D, X 15 is selected from amino acid residues Y, F, X 16 is selected from amino acid residues F, Y.
In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises:
(a) CDR1 having a sequence as set forth in any one of SEQ ID NOs 17, 1, 5, 9, 13 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) as compared to the sequence set forth in any one of SEQ ID NOs 17, 1, 5, 9, 13;
(b) CDR2 having a sequence as set forth in any one of SEQ ID NOs 18, 2, 6, 10, 14 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) as compared to the sequence set forth in any one of SEQ ID NOs 18, 2, 6, 10, 14, and
(C) CDR3 having a sequence as set forth in any one of SEQ ID NOs 19, 3, 7, 11, 15 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1,2 or 3 amino acid substitutions, deletions or additions) as compared to the sequence set forth in any one of SEQ ID NOs 19, 3, 7, 11, 15.
In certain embodiments, the substitution is a conservative substitution.
In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises:
(1) CDR1 as shown in SEQ ID NO. 17, CDR2 as shown in SEQ ID NO. 18, and CDR3 as shown in SEQ ID NO. 19;
(2) CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO. 3;
(3) CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO. 7;
(4) CDR1 as shown in SEQ ID NO. 9, CDR2 as shown in SEQ ID NO. 10, and CDR3 as shown in SEQ ID NO. 11;
Or alternatively
(5) CDR1 as shown in SEQ ID NO. 13, CDR2 as shown in SEQ ID NO. 14, and CDR3 as shown in SEQ ID NO. 15.
In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of:
(i) A sequence as set forth in any one of SEQ ID NOs 20, 4, 8, 12, 16;
(ii) A sequence having one or more amino acid substitutions, deletions or additions (e.g.1, 2, 3,4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in any one of SEQ ID NOs 20, 4, 8, 12, 16, or
(Iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs 20, 4, 8, 12, 16.
In certain embodiments, the substitution is a conservative substitution.
In certain embodiments, the single domain antibody or antigen binding fragment thereof is capable of specifically binding to hGH in a first solvent to form a complex, and the single domain antibody or antigen binding fragment thereof is capable of dissociating in a second solvent to form a complex with hGH;
Wherein the pH of the first solvent is in the range of 6.5-8.5 (e.g., 7.2-7.6) and the pH of the second solvent is in the range of 2.5-3.5 (e.g., 2.8-3.2).
In certain embodiments, the first solvent is phosphate buffer, tris-HCl buffer, HEPES buffer at a pH of 6.5-8.5 (e.g., 7.2-7.6).
In certain embodiments, the second solvent is a citrate buffer, gly-HCl buffer, acetate buffer at a pH of 2.5-3.5 (e.g., 2.8-3.2).
Isolated nucleic acid molecules
In another aspect, the application also provides an isolated nucleic acid molecule encoding a single domain antibody or antigen binding fragment thereof as described above.
Carrier body
In another aspect, the application also provides a vector comprising a nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.
Host cells
In another aspect, the application also provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E.coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.
Preparation method
The single domain antibodies or polypeptide constructs or fusion proteins of the invention may be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the single domain antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transformed/transfected. The transformed/transfected host cells are then cultured under specific conditions and express the single domain antibodies of the invention.
Antigen binding fragments of the invention may be obtained by hydrolysis of intact nanobody molecules (see Morimoto et al, J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al, science 229:81 (1985)). In addition, these antigen-binding fragments may also be produced (reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999);Little et al.,Immunol.Today,21:364-370(2000)). directly from recombinant host cells, other techniques for preparing these antigen-binding fragments being well known to those of ordinary skill in the art.
In another aspect, the application also provides a method of preparing a single domain antibody or antigen-binding fragment thereof as described above, comprising culturing a host cell as described above under conditions that allow expression of the protein, and recovering the single domain antibody or antigen-binding fragment thereof from the cultured host cell culture.
Bispecific or multispecific antibodies
In another aspect, the application also provides a bispecific or multispecific antibody comprising a single domain antibody, or antigen-binding fragment thereof, as described above.
In certain embodiments, the bispecific or multispecific antibody specifically binds hGH, and additionally specifically binds one or more other targets.
In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.
The application also provides application of the single domain antibody or the antigen binding fragment thereof in hGH purification or detection. In a first aspect, the present application provides the use of said single domain antibody or antigen binding fragment thereof in the purification of hGH.
Conjugate(s)
In one aspect, the application also provides a conjugate comprising a single domain antibody or antigen binding fragment thereof as described above, and a solid support linked to the single domain antibody or antigen binding fragment thereof.
In certain embodiments, the solid support is selected from the group consisting of magnetic beads, agarose microspheres, dextran microspheres, polymethacrylates, polystyrene, silica gel microspheres, and combinations thereof.
In certain embodiments, the solid support is selected from porous materials. For example, the solid support is selected from one porous material or a combination of porous materials.
In certain embodiments, the conjugate is capable of specifically binding to hGH to form a complex in a first solvent, and the complex formed by the conjugate and hGH is capable of dissociating in a second solvent.
In certain embodiments, the first solvent and/or the second solvent are as defined above.
Method for purifying hGH
In another aspect, the application also provides a method of purifying hGH comprising the use of a conjugate as described above.
In certain embodiments, the method is a method comprising purifying hGH by affinity chromatography, comprising the steps of:
(1) Combining said conjugate with said hGH in a first solvent to form a complex;
(2) Dissociating the complex in a second solvent, and,
(3) Collecting the dissociation product containing said hGH;
wherein the first solvent and the second solvent are as defined above.
In another aspect, the application also provides the use of a single domain antibody or antigen binding fragment or conjugate thereof as described above in the preparation of a hGH purification reagent.
In a second aspect, the application provides the use of said single domain antibody or antigen binding fragment thereof in the detection of hGH.
Conjugate(s)
In one aspect, the application also provides a conjugate comprising a single domain antibody or antigen binding fragment thereof as described above, and a detectable label linked to the single domain antibody or antigen binding fragment thereof.
In certain embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.
In another aspect, the application also provides a method for detecting the presence or level of hGH in a sample, comprising the use of a single domain antibody or antigen binding fragment or conjugate thereof as described above.
In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.
In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.
In certain embodiments, the method comprises using a conjugate as described above.
In certain embodiments, the method comprises using a single domain antibody or antigen binding fragment thereof as described above, and the method further comprises detecting the single domain antibody or antigen binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine esters, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).
In certain embodiments, the method comprises (1) contacting the sample with a single domain antibody of the invention, or an antigen binding fragment or conjugate thereof, (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of hGH.
The application also provides a method of diagnosing a disease associated with aberrant levels of hGH comprising detecting levels of hGH in a sample from a subject using a method as described above. In certain embodiments, when the level of hGH in a sample from a subject is significantly increased or decreased as compared to a reference level (e.g., as compared to a healthy control), the subject is indicated to have a disease associated with abnormally higher or lower levels of hGH (e.g., giant/dwarfism).
In a further aspect, the present application also provides the use of a single domain antibody or antigen binding fragment or conjugate thereof as described above for the preparation of a detection reagent for detecting the presence or level of hGH in a sample and/or for diagnosing a disease associated with abnormal hGH levels.
In certain embodiments, the detection reagent detects the presence or level of hGH in a sample by a method as described above.
Kit for detecting a substance in a sample
In a further aspect, the application also provides a kit comprising a single domain antibody or antigen binding fragment thereof as described above, a conjugate as described in the first aspect or a conjugate as described in the second aspect.
In certain embodiments, the kit comprises a conjugate as described in the first aspect.
In certain embodiments, the kit comprises a conjugate as described in the second aspect.
In certain embodiments, the kit comprises a single domain antibody or antigen binding fragment thereof as described above, and a second antibody that specifically recognizes the single domain antibody or antigen binding fragment thereof, optionally the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine esters, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.
In certain embodiments, the kit further comprises a buffer (e.g., a detection buffer, a protein purification buffer).
In certain embodiments, the kit comprises a single domain antibody or antigen binding fragment thereof as described above or a conjugate as described in the first aspect, and a buffer for protein purification.
In certain embodiments, the kit comprises a single domain antibody or antigen binding fragment thereof as described above or a conjugate as described in the second aspect, and a buffer for detection.
Definition of terms
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
As used herein, the term "single domain antibody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region), typically derived from a variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity. Single domain antibodies, also known as nanobodies, are used interchangeably.
As used herein, the term "antigen-binding fragment" of a single domain antibody refers to a polypeptide comprising a fragment of a single domain antibody that retains the ability to specifically bind to the same antigen to which the single domain antibody binds, and/or competes with the single domain antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. In some embodiments, the "antigen binding fragment" of the single domain antibody may be truncated at the N-terminus or C-terminus compared to a full length single domain antibody so that it comprises only a portion of FR1 and/or FR4, or lacks one or both of those framework regions, so long as it substantially retains antigen binding and specificity.
Antigen binding fragments of a single domain antibody (e.g., a nanobody provided by the invention) can be obtained from a given single domain antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen binding fragments of a single domain antibody in the same manner as used for the whole nanobody.
In this context, unless the context clearly indicates otherwise, when referring to the term "single domain antibody" it includes not only whole single domain antibodies, but also antigen binding fragments of single domain antibodies.
As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs are contained in the nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as may be defined in accordance with the Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (LEFRANC ET al., dev. Comparat. Immunol.27:55-77,2003). For a given nanobody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see, for example, LEFRANC ET al. Dev. Comparat. Immunol.27:55-77,2003). In this context, the CDRs of the nanobody are preferably determined by the Kabat numbering system.
As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, the two sequences are the same length.
Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, proc.Natl. Acad. Sci.U.S. A.87:2264-2268, as modified in Karlin and Altschul,1993, proc.Natl. Acad. Sci.U.S. A.90:5873-5877. Such an algorithm was integrated into the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403.
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (K D) of the interaction. In the present invention, the term "K D" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.
The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see MALMQVIST M, nature,1993, 361:186-187). The kdis/kon ratio is equal to the dissociation constant K D (see Davies et al, annual Rev Biochem,1990; 59:439-473). The K D, kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.
As used herein, a detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H、125I、35S、14 C or 32 P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine esters, luminol and derivatives thereof, ruthenium derivatives such as ruthenium terpyridyl), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.
As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.
As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).
The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., ,Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),, incorporated by reference herein. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
Advantageous effects of the invention
The serial single domain antibodies of the anti-human growth hormone provided by the application have high binding activity with human growth hormone. In particular, the single domain antibody of the application has excellent high temperature resistance, acid and alkali resistance and excellent stability, can effectively combine with hGH under neutral condition, and can effectively dissociate with hGH under low pH acid condition, thus being advantageously applied to affinity purification of human growth hormone. In addition, the single domain antibody has the characteristics of small molecular weight, easy production and the like.
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.
FIG. 1 shows the results of an ELISA assay for detecting the binding of 5 single domain antibodies (anti-hGH Nb-1/9/18/20/27) to hGH.
FIG. 2 shows the results of ELISA assays for binding of 3 single domain antibodies (anti-hGH Nb-1/9/18) to hGH after treatment at different temperatures (45 ℃ and 55 ℃) for different times (24 h and 48 h), wherein FIG. 2A shows the results of anti-hGH Nb-1 binding to hGH and FIG. 2B shows the results of anti-hGH Nb-9/18 binding to hGH.
FIG. 3 shows the results of an ELISA assay for binding of 3 single domain antibodies (anti-hGH Nb-1/9/18) to hGH after treatment at pH3.0 for various periods of time (0 h, 24h, 48 h).
FIG. 4 shows the non-reducing SDS-PAGE result of the anti-hGH single domain antibody prepared in example 4, wherein FIG. 4A is the SDS-PAGE result of anti-hGH Nb-1/9/18, lane M is the molecular weight Marker, lane 1 is anti-hGH Nb-18, lane 2 is anti-hGH Nb-9, lane 3 is anti-hGH Nb-1, FIG. 4B is the SDS-PAGE result of anti-hGH Nb-20/27, lane M is the molecular weight Marker, lane 1 is anti-hGH Nb-20, and lane 2 is anti-hGH Nb-27.
FIG. 5 shows a graph of the flow-through rate versus loading of affinity media coupled to different single domain antibodies (anti-hGH Nb-1/9/18/20/27), wherein FIG. 5A is a graph of the flow-through rate versus loading of affinity media coupled to anti-hGH Nb-1/9/18, C represents the concentration of a flow-through sample in the abscissa, C0 represents the loading concentration of hGH stock solution, and FIG. 5B is a graph of the flow-through rate versus loading of affinity media coupled to anti-hGH Nb-20/27.
Sequence information
A description of the sequences to which the present application relates is provided in the following table.
TABLE 1 sequence information
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the molecular biology experimental methods and immunoassays used in the present invention are essentially described in J.Sambrook et al, molecular cloning, in laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-programming molecular biology experimental guidelines, 3 rd edition, john Wiley & Sons, inc.,1995, and the use of restriction enzymes is in accordance with conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.
EXAMPLE 1 construction of an anti-recombinant human growth hormone (hGH) single domain antibody immune library
After emulsifying 2mg hGH protein (amino acid sequence shown as SEQ ID NO: 27) with Freund's complete adjuvant, alpaca (Vicugna pacos) was subjected to subcutaneous multipoint injection immunization. The hGH protein was subjected to secondary immunization with 1.5mg of Freund's incomplete adjuvant after 20 days of primary immunization, and then immunization was performed every 14 days, and blood was collected intravenously, serum titers were detected by ELISA, lymphocytes were isolated from peripheral blood after seven days of primary immunization, and total RNA was extracted.
RNA extraction was performed with reference to Invitrogen TRIzol TM Reagent instructions. The first strand cDNA was synthesized using total RNA as template, oligo dT and Random 6 as primers, and referring to TakaRa's reverse transcription kit instructions. The variable region encoding genes of heavy chain antibodies were obtained by nested PCR using the 2×q5 high fidelity polymerase of NEB (primers see table 2).
TABLE 2 primer names and sequencesNote that r=a/G, y=c/T, s=c/G.
The first round of PCR amplified cDNA with primers Alpaca-CALL01-F and Alpaca-CALL02-R, respectively, at 98℃for 30s, 98℃for 5s,58℃for 15s,72℃for 20s,27 cycles, and 72℃for 2min.
The first round of PCR products were electrophoresed on a 1.5% (w/v) agarose gel, and about 750bp DNA fragments were recovered as templates for the second round of PCR, and the second round of PCR was performed using primers (Alpaca-IGHV 3S53/61-F, alpaca-IGHV3-3-F, alpaca-IGHV3S59/67-F, alpaca-IGHV3S64/64-F, alpaca-IGHV3S65-F equimolar mix as forward primers, alpaca-IGHJ1-R, alpaca-IGHJ2/3/7-R, alpaca-IGHJ5-Rnew, alpaca-IGHJ4/6-R equimolar mix as reverse primers) under conditions of 98 ℃,30S, 98 ℃,5S,58 ℃,15S,72 ℃,20S,27 cycles, and extension for 2min. Recovered and quantified by using an Omega gel recovery kit, and stored at-20 ℃ for later use. And (3) cutting the phagemid vector pGS249-3 and the PCR recovery product by BglI, cutting the phagemid vector pGS249-3 by BglI, cutting by NsiI to avoid self-connection of the vectors, passing through a column for recovery and quantification by a gel recovery kit, and connecting for 1 hour at 22 ℃ in a molar ratio of 1:3 to obtain a connection product.
After the connection product is recovered by a gel recovery kit, the connection product is dissolved in 50 mu l of ultrapure water, 500ng of the purified connection product is electrically transferred to competent cells of escherichia coli TG1, 16 branches are electrically transferred, a 37 ℃ preheated SOC culture medium is immediately added after the electric transfer, after the mixture is uniformly mixed, a 37 ℃ shaking table is used for recovering for 1 hour, 50 mu l of bacterial liquid is taken for each branch after the recovery, the bacterial liquid is mixed in a gradient manner, a 2 XYT plate (containing 100 mu g/ml of ampicillin) is coated, the rest bacterial liquid is completely coated on a 150X 20mm 2 XYT plate (containing 100 mu g/ml of ampicillin), and the bacterial liquid is reversely cultured overnight at 37 ℃. The following day colony PCR was performed using M13R (SEQ ID NO: 21) and 249R (SEQ ID NO: 22) primers, and the library capacity and library positive rate were calculated. The lawn on the plate was scraped with 2 XYT medium (containing 100. Mu.g/ml ampicillin), and then glycerol was added at a final concentration of 15% (V/V), and the mixture was sub-packaged and stored at-80℃for further use.
According to the calculated storage capacity result, a bacterial solution with 100 times of storage capacity is inoculated in 10L of 2 XYT culture medium (containing 2% (w/v) glucose and 100 mu g/ml ampicillin), cultured at 37 ℃ and 200rpm until OD 600 =0.5, helper phage M13KO7 is added according to the infection complex number of 100:1, the culture is carried out at 37 ℃, standing and incubation is carried out for 15min, and then shaking culture is carried out at 37 ℃ and 200rpm for 1 hour. The culture was centrifuged and the pellet was resuspended in 5L of 2 XYT (containing 100. Mu.g/ml ampicillin and 70. Mu.g/ml kanamycin) and incubated overnight at 30℃with shaking at 200 rpm. Centrifuging to collect supernatant, adding 1/4 volume of PEG/NaCl into the supernatant, mixing, ice-bathing for 1 hr, centrifuging, re-suspending the precipitate with PBS to obtain anti-hGH single domain antibody immune library, taking 20 μl of measured titer, adding glycerol with final concentration of 50% (V/V), sub-packaging, and storing at-80deg.C.
Example 2 panning and identification of anti-hGH Single Domain antibodies
The anti-hGH single-domain antibody immune library obtained in example 1 was panned against hGH using a solid phase panning method. 1ml of hGH protein was added to the immune tubes, coated overnight at 4℃and each round of panning had a hGH coating concentration of 20. Mu.g/ml, 10. Mu.g/ml, respectively, one blank of immune tube (as control) was taken, 5% (w/V) mill was added, blocked at 37℃for 2h, PBST (containing 0.05% (V/V) Tween-20) was added to wash 3 times, phage (5X 10 13 pfu) from which 5% mill had been bound was removed was added to each of the 2 immune tubes, incubated for 1h at room temperature, washed 10 times with PBST (15 times for the latter 2 rounds), unbound phage was washed off, phage adsorbed on the immune tubes was eluted with 0.02M citric ACID-sodium citrate (ACID, pH 3.0), 1. Mu.l of 1M Tris-HCl (pH 8.0) was used to neutralize the eluate, 100. Mu.l of OD 600 = 0.5, the remaining titer was determined, and the eluate was used for the next round of amplification.
After four rounds of panning, the monoclonal was picked, cultured overnight at 37 ℃ and then transferred to allow the bacterial solution to grow to OD 600 =0.5, induced overnight with IPTG 30 ℃ at a final concentration of 1mM, and the supernatant was collected by centrifugation for ELISA experiments. 1 μg/ml hGH was coated overnight at 4 ℃, blocked at 5% milk,37 ℃ for 2h, washed 3 times with PBST, 50 μl of induction supernatant was added, washed 3 times with PBST, anti-His-HRP secondary antibody was added, and developed.
ELISA positive clones were sent to Jilin provincial treasury Mei Biotechnology Co.Ltd for sequencing, and after sequence analysis, 5 single domain antibodies with good diversity were selected for subsequent eukaryotic expression.
Example 3 preparation of anti-hGH Single-Domain antibodies and physicochemical screening
3.1 Preparation of anti-hGH Single-Domain antibodies and ELISA binding experiments
Cloning the 5 single domain antibody gene fragments into eukaryotic expression vector pGS003, and successfully constructing the expression vector by sequencing.
Plasmids were extracted and transient expression was performed in FreeStyle medium using FreeStyle TM 293E cells. 24 hours prior to transfection, 25ml of 293E cells at 0.5X10 6 cells/ml were inoculated in 125ml Erlenmeyer flasks and shake-cultured at 130rpm in a 37℃5% CO 2 incubator. At the time of transfection, 60. Mu.l of 293E Fectin was added to 1ml of Opti-MEM, and after thorough mixing, incubated at room temperature for 5 minutes while 25. Mu.g of the total plasmid DNA of the recombinant vector was dissolved in 1ml of Opti-MEM. The plasmid DNA and 293E Fectin were then thoroughly mixed and incubated at room temperature for 15 minutes in a total volume of 2ml, and the mixture was then added to the cell culture wells in total, mixed well and shake-incubated in a 37℃5% CO 2 incubator at 130rpm for 7 days. And (3) centrifuging the culture solution at a high speed, and taking the supernatant to perform vacuum filtration through a microporous filter membrane. And (3) purifying by using a nickel column according to an operation method provided by a manufacturer to obtain the purified single domain antibody.
Coating 0.5. Mu.g/ml hGH (amino acid sequence as SEQ ID NO: 27) 50. Mu.l/well, overnight at 4 ℃, blocking with 250. Mu.l/well 1% (w/v) BSA, then washing 3 times with PBST, starting with 10. Mu.g/ml, three-fold dilution, total of 8 spots of purified Anti-hGH single domain antibody prepared as above, 50. Mu.l/well, incubation at room temperature for 1h, PBST, washing 3 times, addition of Anti-His-HRP secondary antibody, development.
The results are shown in FIG. 1, which shows that the 5 single domain antibodies (anti-hGH Nb-1/9/18/20/27) all have higher binding activity with hGH.
The affinity purification procedure was simulated to verify that the antibody molecule was able to dissociate normally under 0.02M ACID of citric ACID-sodium citrate (pH 3.0). The procedure was as follows, coating with 0.5. Mu.g/ml hGH, blocking with 250. Mu.l/well 1% (w/v) BSA overnight, then washing 3 times with PBST, adding 10. Mu.g/ml Anti-hGH single domain antibody, 50. Mu.l/well, incubating for 1h at room temperature, washing with ACID, washing with PBST (pH 7.2-7.4) as control, finally adding Anti-His-HRP secondary antibody, developing, and the results are shown in Table 3.
TABLE 3 Table 3
The results show that the single domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, anti-hGH Nb-27 bind well to hGH under neutral conditions and dissociate well from hGH under low pH acidic conditions.
3.2 Octet assay for affinity of hGH with anti-hGH single domain antibodies
The equilibration buffer PBS, 30. Mu.g/ml anti-hGH single domain antibody and hGH were first added to the microplate. First baseline adjustment, HIS1K probe was used to bind to 30. Mu.g/ml anti-hGH single domain antibody. Baseline adjustment is then performed again, binding to hGH with a probe conjugated to an anti-hGH single domain antibody. Affinity (M) of hGH to anti-hGH single domain antibody was obtained using data analysis software. The results are shown in Table 4.
Table 4 affinity of single domain antibodies to hGH
3.3 Evaluation of stability of anti-hGH Single Domain antibodies
Each 4 tubes of 100. Mu.g/tube of purified single domain antibody (anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18) was placed in a water bath at 45℃and 55℃for 24 hours, 48 hours, respectively, and ELISA experiments were performed according to the above step 3.1 to verify whether the anti-hGH single domain antibody was stable at high temperature. Specific results are shown in fig. 2A and fig. 2B, and the results show that the 3 single domain antibodies keep the binding activity with hGH after the above treatment, and have better stability.
Each 3 tubes of 100. Mu.g/tube purified single domain antibody (anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18) was placed at pH3.0 for 24h, 48h, respectively, and ELISA experiments were performed to verify whether the anti-hGH single domain antibody was stable under acidic conditions. The specific results are shown in figure 3, and the results show that the 3 single domain antibodies keep the binding activity with hGH after the treatment, and have better stability.
EXAMPLE 4 preparation and purification of anti-hGH Single-domain antibodies
Antibody preparation purification was performed on the single domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20, anti-hGH Nb-27.
4.1 Preparation and purification of anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18
Capturing target protein by ProA affinity chromatography, and specifically preparing the anti-hGH Nb-9 by the process and parameters shown in Table 5-1.
TABLE 5-1 ProA affinity chromatography procedure and parameters
Capturing target proteins through Ni affinity chromatography, and specifically preparing the anti-hGH Nb-18 and anti-hGH Nb-1 by the following steps and parameters in Table 5-2.
TABLE 5-2 Ni affinity chromatography procedure and parameters
The purity of the prepared hGH single-domain antibody candidate molecule was evaluated by SDS-PAGE, the result of which is shown in FIG. 4A, and the preparation collection information of each purified single-domain antibody sample is shown in Table 6.
TABLE 6 sample preparation collection information
According to the analysis of electrophoresis results of the sample collected by candidate molecule preparation, the purity of 3 candidate molecules is more than 95%, and the method can be used for continuing the preparation experiment of the affinity coupling medium.
4.2 Preparation and purification of anti-hGH Nb-20 and anti-hGH Nb-27
The target proteins are captured through Ni affinity chromatography, and specific preparation flows and parameters of the anti-hGH Nb-20 and the anti-hGH Nb-27 are shown in Table 7.
TABLE 7 Ni affinity chromatography procedure and parameters
The purity of the prepared hGH single-domain antibody candidate molecule was evaluated by SDS-PAGE, the result of which is shown in FIG. 4B, and the preparation collection information of each purified single-domain antibody sample is shown in Table 8.
Table 8 sample preparation collection information
According to the analysis of electrophoresis results of the sample collected by candidate molecule preparation, the purity of 2 candidate molecules is more than 95%, and the method can be used for continuing the preparation experiment of the affinity coupling medium.
Example 5 preparation of anti-hGH Single-domain antibody affinity coupling Medium
Affinity coupling medium preparation is carried out on the single domain antibodies anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18, anti-hGH Nb-20 and anti-hGH Nb-27.
5.1 Preparation of anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18 affinity coupling Medium
5.1.1 Medium swelling
1.5G of dry medium of candidate molecules anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18 purified as in example 4 was weighed, 10ml of 1mM HCl (pH 3.0) was added to each, and mixed in contact thoroughly at room temperature with appropriate shaking, swelled for 2 hours, the medium was poured into a solid phase extraction column, the supernatant was discarded, and washed with 1mM HCl.
5.1.2 Coupling
And (3) flushing the medium in the solid phase extraction column by using a coupling buffer solution of 0.5M NaCl-0.1M NaHCO 3 (pH 7.7-8.3) until the pH of the outflow end is consistent with that of the coupling buffer solution, and then pumping the liquid. Pouring the treated medium into a sterile centrifuge tube, pre-cooling for 0.5h at 4 ℃, replacing each hGH single domain antibody into a coupling buffer solution, referencing the medium dosage of 1-10 mu mol/ml, ensuring that the total amount of each hGH single domain antibody molecule is consistent, and pouring into the centrifuge tube and uniformly mixing with the medium. Shake at room temperature for 2h. The coupled medium was then poured into a solid phase extraction column, the coupled supernatant was collected, the medium was rinsed with 0.5CV coupling buffer, and the rinses were combined. Protein concentration was measured by uv to calculate the coupling efficiency of the protein.
5.1.3 Closure
The affinity coupling medium was blocked with 1M Tris-HCl-100mM EDTA (pH 7.8-8.2) at 4℃overnight.
5.1.4 Elution
Using elution solution a:0.5M NaCl-0.1M Tris-HCl-10mM EDTA (pH 7.7-8.3), eluting solution B, 20mM citrate (pH 3.0) and regeneration solution C, 1M acetic acid, sequentially flushing 3 times of the volume, collecting eluting (regeneration) peak, and detecting the protein concentration by ultraviolet. Repeating for three times. The coupling ratio of the affinity coupling medium was calculated.
Specific information on candidate molecule affinity coupling experiments is shown in Table 9.
TABLE 9 hGH Single-domain antibody candidate molecule coupled affinity Medium information
The coupling ratios of the candidate molecules anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 as ligands to CNBr activated Sepharose B medium were 15.92mg/ml, 17.13mg/ml and 15.83mg/ml, respectively.
5.2 Preparation of anti-hGH Nb-20 and anti-hGH Nb-27 affinity coupling Medium
5.2.1 Medium swelling
The candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 purified as in example 4 were weighed 10g and 2g of dry medium respectively, 1mM HCl (pH 3.0) was added to each of them in a ratio of 1:5 (w/v), and the mixture was thoroughly mixed by shaking at room temperature, swelled for 2 hours, the medium was poured into a solid phase extraction column, the supernatant was discarded, and washing was performed with 1mM HCl.
5.2.2 Coupling
And (3) flushing the medium in the solid phase extraction column by using a coupling buffer solution of 0.5M NaCl-0.1M NaHCO 3 (pH 7.7-8.3) until the pH of the outflow end is consistent with that of the coupling buffer solution, and then pumping the liquid. Pouring the treated medium into a sterile centrifuge tube, pre-cooling for 0.5h at 4 ℃, replacing each hGH single domain antibody into a coupling buffer solution, referencing the medium dosage of 1-10 mu mol/ml, ensuring that the total amount of each hGH single domain antibody molecule is consistent, and pouring into the centrifuge tube and uniformly mixing with the medium. Shake overnight at 2-8 ℃ for 15h. The coupled medium was then poured into a solid phase extraction column, the coupled supernatant was collected, the medium was rinsed with 0.5CV coupling buffer, and the rinses were combined. Protein concentration was measured by uv to calculate the coupling efficiency of the protein.
5.2.3 Closure
The affinity coupling medium is blocked with 1M Tris-HCl-100mM EDTA (pH 7.8-8.2), at 4℃for at least 3h.
5.2.4 Medium flushing
The protein concentration was detected by sequentially washing 3 volumes with a coupling equilibration solution 1:0.5M NaCl-0.1M Tris-HCl-10mM EDTA (pH 7.7-8.3), an elution solution 2:20mM citrate (pH 3.0) and a regeneration solution 3:1M acetic acid, and collecting the elution (regeneration) peaks. Repeating for three times. The coupling ratio of the affinity coupling medium was calculated.
Specific information on candidate molecule affinity coupling experiments is shown in Table 10.
TABLE 10 hGH Single-domain antibody candidate molecule coupled affinity Medium information
The coupling ratio of candidate molecules anti-hGH Nb-20, anti-hGH Nb-27 as ligand to CNBr activated Sepharose B medium was 14.51mg/ml and 16.18mg/ml, respectively.
EXAMPLE 6 evaluation of affinity coupling Medium for anti-hGH Single-domain antibodies
6.1 Evaluation of anti-hGH Nb-1, anti-hGH Nb-9, anti-hGH Nb-18 affinity coupling Medium
The conjugated hGH single domain antibody affinity medium was loaded into Bognon 10/20 chromatography columns, all 3.14ml column volumes. The affinity coupling medium of the different ligands was evaluated by dynamic binding capacity (Dynamic Binding Capacity, DBC), efficient elution and purification effect (purity, related proteins, etc.).
6.1.1 Dynamic Loading determination of affinity Medium
Referring to the operational procedures and parameters of Table 11, under the condition of retention time of 10min, hGH stock solution (amino acid sequence shown as SEQ ID NO:27; loading after dilution, concentration 1.33mg/ml after dilution) was overloaded (20 mg/ml), flow-through solution was collected and the concentration was determined, and the dynamic binding capacity at 5% flow-through rate was calculated.
TABLE 11 affinity chromatography load determination procedure and parameters
The flow-through sample concentration was detected, and the flow-through rate was calculated, thereby obtaining the maximum binding capacity corresponding to the retention time at 5% of the sample flow-through rate (see table 12). The affinity medium flow-through versus loading for the coupling of different anti-hGH single domain antibodies is shown in FIG. 5A.
Table 12 maximum binding capacity corresponding to retention time of 10min at 5% sample flow rate
According to the result, the retention time of the candidate molecule anti-hGH Nb-18 affinity medium at the sample flow rate of 5% for 10min corresponds to a maximum binding capacity of 7.24mg/ml, and the maximum dynamic binding capacities of the anti-hGH Nb-9 affinity medium and the anti-hGH Nb-1 affinity medium are respectively 4.65mg/ml and 6.30mg/ml.
6.1.2 Evaluation of efficient elution of affinity Medium samples
Referring to the operational procedures and parameters shown in Table 13, different candidate molecule affinity media were loaded at their maximum binding capacity, the loaded sample was hGH stock solution (stock solution was loaded after dilution, concentration after dilution was 1.33 mg/ml), and the sample product yield was collected after detection purification to evaluate the effective elution profile of the affinity coupling media.
TABLE 13 efficient elution procedure and parameters for affinity chromatography
The collected sample concentration was measured, and the yield of hGH sample eluted from each candidate molecule affinity medium at pH3.0 was calculated in combination with the information such as the collected volume, and the results are shown in Table 14.
TABLE 14 affinity chromatography product yield Table
From the above results, it was shown that the candidate molecules anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 affinity media were eluted under such severe conditions as pH3.0, with yields of 85.68%, 86.99% and 70.17%, respectively, for hGH.
6.1.3 Evaluation of affinity Medium sample purification Effect
Referring to the preparation flow and parameters of Table 15, chromatographic procedures were performed over the loading ranges of the different candidate molecules, and the purified hGH was tested for product quality by loading with hGH E.coli lysate supernatant (1.26 mg/ml supernatant concentration as determined by SDS-PAGE gray scale) to evaluate the purification effect of the affinity coupling medium. The assay items were SEC (size exclusion chromatograghy, size exclusion chromatography) purity of hGH and content of related proteins (non-hGH proteins).
The concentration of the affinity-collected samples was determined and the SEC purity of hGH and the content of related proteins (other than hGH proteins) in the collected samples were measured, and the results are shown in Table 15, in which the SEC purity of hGH was measured by SEC-HPLC and the content of related proteins was measured by RP-HPLC.
TABLE 15 quality of hGH product from affinity chromatography collection
As can be seen from the evaluation results of the purification effect in Table 15, the purities of the hGH samples purified by the anti-hGH Nb-18, anti-hGH Nb-9 and anti-hGH Nb-1 candidate molecule affinity coupling medium can reach 94.58%, 92.40% and 87.96%, respectively, in terms of SEC purity, and the relevant protein contents are 12.17%, 12.59% and 14.94%, respectively, in terms of impurities.
The dynamic loading, effective elution and purification effect evaluation results of the affinity media of different candidate molecules under the experimental conditions are synthesized, and all three candidate molecule affinity media are good in performance and can be used for subsequent production processes.
6.2 Evaluation of anti-hGH Nb-20, anti-hGH Nb-27 affinity coupling Medium
The conjugated hGH single domain antibody affinity medium was loaded into a Bognon 10/20 column, each 6.12ml column volume. The affinity coupling medium of the different ligands was evaluated by dynamic binding capacity (Dynamic Binding Capacity, DBC), efficient elution and purification effect (purity, related proteins, etc.).
6.2.1 Dynamic Loading determination of affinity Medium
Referring to the operational procedures and parameters of Table 16, under the condition of keeping the time for 5min, the hGH stock solution (amino acid sequence shown as SEQ ID NO:27; loading after dilution, concentration 1-1.5mg/ml after dilution) was overloaded (20 mg/ml) and loaded, the flow-through solution was collected and the concentration was measured, and the dynamic binding capacity at 10% flow-through rate was calculated.
Table 16 affinity chromatography load determination procedure and parameters
The flow-through sample concentration was detected, and the flow-through rate was calculated, thereby obtaining the maximum binding capacity corresponding to the retention time at 10% of the sample flow-through rate (see table 17). The affinity medium flow-through versus loading for the coupling of different anti-hGH single domain antibodies is shown in FIG. 5B.
Table 17 maximum binding capacity corresponding to retention time 5min at 10% sample flow rate
According to the results, the maximum binding capacity of candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 affinity media, which correspond to a retention time of 5min at 10% sample flow rate, are respectively 10.04mg/ml and 11.15mg/ml.
6.2.2 Evaluation of efficient elution of affinity Medium samples
Referring to the operational procedures and parameters shown in Table 18, different candidate molecule affinity media were loaded at their maximum binding capacity, the loaded samples were intermediate products in the production process of hGH stock solution, and the yield of the sample products was collected after detection and purification to evaluate the purification effect of the affinity coupling media.
Table 18 affinity coupling Medium purification Effect evaluation procedure and parameters
The collected sample concentration was measured, and the yield of hGH sample eluted from each candidate molecule affinity medium at pH3.0 was calculated in combination with the information such as the collected volume, and the results are shown in Table 19.
TABLE 19 affinity chromatography product yield Table
The results show that the candidate molecules anti-hGH Nb-20 and anti-hGH Nb-27 affinity media have yields of 93.48% and 95.98% for hGH, respectively, when eluted under the harsher conditions of pH 3.0.
6.2.3 Evaluation of affinity Medium sample purification Effect
Referring to the preparation flow and parameters of Table 20, chromatographic procedures were performed over the loading ranges of the different candidate molecules, and the purified hGH was tested for product quality by loading with hGH E.coli lysate supernatant (1.26 mg/ml supernatant concentration as determined by SDS-PAGE gray scale) to evaluate the purification effect of the affinity coupling medium. The assay items were SEC (size exclusion chromatograghy, size exclusion chromatography) purity of hGH and content of related proteins (non-hGH proteins).
The concentration of the affinity-collected samples was determined and the SEC purity of hGH and the content of related proteins (other than hGH proteins) in the collected samples were measured, and the results are shown in Table 20, in which the SEC purity of hGH was measured by SEC-HPLC and the content of related proteins was measured by RP-HPLC.
TABLE 20 quality of hGH product collected by affinity chromatography
As can be seen from the evaluation results of the purification effect in Table 20, the purities of the purified hGH samples by the anti-hGH Nb-20 and anti-hGH Nb-27 candidate molecule affinity coupling medium respectively reached 99.84% and 99.39% in terms of SEC purity, and the relevant protein contents were 7.33% and 7.32% in terms of impurities, respectively.
Comprehensive dynamic loading, effective elution and purification effect evaluation results of affinity media of different candidate molecules under the experimental conditions, and the affinity media of the two candidate molecules are good in performance and can be used for subsequent production processes.
While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.
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