WO2022131788A1 - Procédé d'immunoprécipitation utilisant une protéine de liaison à un anticorps et une protéine de fusion de calséquestrine - Google Patents

Procédé d'immunoprécipitation utilisant une protéine de liaison à un anticorps et une protéine de fusion de calséquestrine Download PDF

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WO2022131788A1
WO2022131788A1 PCT/KR2021/019070 KR2021019070W WO2022131788A1 WO 2022131788 A1 WO2022131788 A1 WO 2022131788A1 KR 2021019070 W KR2021019070 W KR 2021019070W WO 2022131788 A1 WO2022131788 A1 WO 2022131788A1
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protein
glu
antibody
asp
leu
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김성현
박희선
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한국세라믹기술원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the use of an antibody-binding protein and a calciquestrin fusion protein, and specifically, an antibody-binding protein and a calciquestrin fusion protein for immunoprecipitation, and immunoprecipitation using the antibody-binding protein and calciquestrin fusion protein It relates to a kit for use, and an immunoprecipitation method using an antibody binding protein and a calciquestrin fusion protein.
  • Immunoprecipitation is a technique for separating an antigen (or a protein having affinity for an antigen) from a solution using the specific immune reactivity of an antigen and an antibody. Determination of the molecular weight of the antigen, and protein interaction It is widely used for research, measurement of specific enzymatic activity, monitoring of post-transcriptional changes in protein, and confirming the amount and presence of protein. In order to confirm the protein-protein interaction in the immunoprecipitation method, the role of a precipitating agent that can effectively detect the protein-bound antibody is essential.
  • Protein A the binding partner of the antibody, is a cell surface protein found in the cell wall of Staphylococcus aureus with a size of 42 kDa.
  • the main function of this protein is that, before the antibody recognizes the antigen of the microorganism and binds to the receptor of the phagocyte, the protein A (Protein A) first binds to the Fc portion of the antibody to avoid the immune attack of the host cell, so that the antibody-receptor It is to avoid the immune-induced response by interfering with the reaction of the liver.
  • This protein consists of five homologous domains and forms an alpha helix structure. In addition, each domain has properties of binding to different immunoglobulins.
  • Protein silica forming G (Protein G) and protein L (Protein L) as binding partners having binding ability to immunoglobulin as well as protein A (Protein A) were also discovered and reported.
  • protein A (Protein A), protein G (Protein G), or a mixture of A/G is mainly produced and commercialized.
  • these products have a limitation in performing efficient immunoprecipitation due to the small amount of extracted protein compared to the amount used.
  • agarose beads used as a sedimentation agent for existing products have high non-specific adsorption, so there is a limit to sedimentation of unwanted proteins.
  • An object of the present invention is to provide an antibody binding protein and a calciquestrin fusion protein for immunoprecipitation.
  • Another object of the present invention is to provide a kit for immunoprecipitation using an antibody binding protein and a calciquestrin fusion protein.
  • Another object of the present invention is to provide an immunoprecipitation method using an antibody binding protein and a calciquestrin fusion protein.
  • fusion protein refers to a protein prepared by joining two or more genes encoded separately from each other.
  • the fusion protein can be artificially prepared through recombinant DNA technology.
  • the fusion protein may be in a form in which a physiologically active protein or peptide and an immunoglobulin Fc fragment are linked without a linker or via a peptidic linker.
  • the peptidic linker refers to a peptide that connects two types of proteins constituting the fusion protein to each other.
  • the peptidic linker may be included so that two proteins, which are components of the fusion protein, can be independently folded in the form of a fusion protein or maintain their respective functions, but is not limited thereto.
  • immunoglobulin Fc fragment refers to the heavy and light chain variable regions of immunoglobulin, heavy chain constant region 2 (CH2) and heavy chain constant region except for heavy chain constant region 1 (CH1) and light chain constant region 1 (CL1). It refers to a region 3 (CH3) portion, and also includes a hinge portion in the heavy chain constant region.
  • immunoglobulin Fc fragment of the present invention has substantially the same or improved effect as that of the native type, part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region except for only the heavy and light chain variable regions of immunoglobulin It may be an extended Fc fragment comprising region 1 (CL1).
  • the immunoglobulin Fc fragment of the present invention includes a native amino acid sequence as well as sequence derivatives thereof.
  • the amino acid sequence derivative means having a sequence different from that of a natural amino acid sequence by one or more amino acid residues, and may be naturally occurring or artificially generated.
  • Fc fragments of immunoglobulins include derivatives by deletion, insertion, non-complementary or complementary substitution, or a combination thereof. Insertions usually consist of a contiguous sequence of about 1 to 20 amino acids, although larger insertions are possible. Deletions typically consist of about 1 to 30 residues.
  • the most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, or the like.
  • Such immunoglobulin Fc fragments may be obtained from natural forms isolated from in vivo animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
  • the method for obtaining from the native type can be obtained by isolating the entire immunoglobulin from a living body of a human or animal, and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab')2.
  • Fc or pF'c may be separated using size-exclusion chromatography or the like.
  • the immunoglobulin Fc fragment according to the present invention may include protein A, protein G, protein A/G or protein L derived from a microorganism.
  • calcium-binding protein is a calcium-binding protein of the sarcoplasmic reticulum, and even though the concentration of calcium in the sarcoplasmic reticulum is higher than that of the cytoplasm, it binds to calcium after muscle contraction Allows storage of calcium ions in the cisterna. Calcium storage capacity is very good because one calciquestrin molecule can bind multiple calcium (eg, each calciquestrin molecule has 40-50 calcium binding sites).
  • antibody-binding protein is a protein having an activity of binding to the Fc region of an antibody, and proteins and peptides having such activity can be used without limitation.
  • the antibody-binding protein may be protein Z, protein G, protein A or protein A/G.
  • Z protein is obtained from protein A of Staphylococcus aureus, which refers to a protein used for defense against antibodies present in host cells.
  • the Z protein is used as a self-defense mechanism that binds to the Fc region of an antibody in a host cell and prevents phagocytosis by macrophages from occurring.
  • a tandem repeat dimer made by manipulating only the antibody-binding site in Protein A is referred to as a "Z domain".
  • protein A includes protein A recovered from its natural source, protein A synthetically produced (e.g., by peptide synthesis, recombinant technology, etc.), and variants thereof having the ability to bind proteins having CH2/CH3 domains. do.
  • protein G refers to a cell wall protein from Group G Streptococcus. Protein G is a type III Fc receptor that binds with high affinity to the Fc region of antibodies, particularly IgG antibodies. As used herein, the term “protein G” refers to protein G recovered from its natural source, protein G synthesized (eg, by peptide synthesis, recombinant technology, etc.), and a protein that binds a protein having an Fc region. and variants thereof with the ability.
  • protein A/G refers to a gene fusion product secreted from a non-pathogenic form of Bacillus as a genetically processed protein combining the IgG binding profile of protein A and protein G.
  • the immunoprecipitation method using the antibody binding protein and the calciquestrin fusion protein according to the present invention can separate antigens and antibodies with higher purity than the conventional ProteinA-Agarose method.
  • FIG. 1 shows a schematic diagram of an immunoprecipitation method using a Z-domain and a calciquestrin fusion protein according to the present invention.
  • Figure 2 shows the Turbidity results according to the calcium concentration of Z-CSQ1 and ZZ-CSQ1 according to Example 2.
  • FIG. 4 shows the results of confirming the calcium precipitation of Z-CSQ1 and ZZ-CSQ1 by concentration.
  • FIG. 5 shows the results of confirming the separation of ZZ-CSQ1 fusion protein and ProteinA-Agarose with Herceptin antibody.
  • the present invention is to provide an antibody binding protein and a calciquestrin fusion protein for immunoprecipitation, wherein the calciquestrin comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 3 or any one of SEQ ID NOs: 1 to 3 consists of one amino acid sequence,
  • the antibody-binding protein is characterized in that it is a Z-domain, protein A, protein G or protein A/G.
  • the antibody-binding protein comprises any one of the amino acid sequences of SEQ ID NOs: 4 to 6 or comprises any one of the amino acid sequences of SEQ ID NOs: 4 to 6 Z-domain consisting of, protein A comprising the amino acid sequence of SEQ ID NO: 7 or consisting of the amino acid sequence of SEQ ID NO: 7, protein G comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or amino acid of SEQ ID NO: 9 It is characterized in that it is protein A/G comprising the sequence or consisting of the amino acid sequence of SEQ ID NO: 9.
  • the antibody is CD3 (eg, OKT3), CD52 (eg, alemtuzumab; Campath®), VEGF (eg, bevacizumab) Avastin®), BSA, EGFR (eg cetuximab; Erbitux®), CD33 (eg gemtuzumab; Mylotarg®), CD20 (eg rituximab; Rituxan®; tositumomab; Bexxar®; ibritumomab; Zevalin ®), HER-2 (eg trastuzumab; Herceptin®), TNF ⁇ (eg adalimumab; Humira®, infliximab; Remicade®; etanercept; Embrel®), CD25 (eg daclizumab; Zenapax®; basiliximab; Simulect®), RSV
  • CD3 eg, OKT3
  • CD52 eg, alemtuzumab;
  • the present invention is to provide a kit for immunoprecipitation using an antibody binding protein and a calciquestrin fusion protein, wherein the calciquestrin comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 3 or SEQ ID NOs: 1 to It consists of an amino acid sequence of any one of 3, and the antibody-binding protein is characterized in that it is a Z-domain, protein A, protein G, or protein A/G.
  • the antibody-binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 4 to 6 or any one of SEQ ID NOs: 4 to 6 Z-domain consisting of the amino acid sequence, protein A comprising the amino acid sequence of SEQ ID NO: 7 or consisting of the amino acid sequence of SEQ ID NO: 7, protein G comprising the amino acid sequence of SEQ ID NO: 8 or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: It is characterized in that it is protein A/G comprising the amino acid sequence of 9 or consisting of the amino acid sequence of SEQ ID NO: 9.
  • the present invention provides an immunoprecipitation method using an antibody binding protein and a calciquestrin fusion protein, the method comprising:
  • A-1) An antigen-antibody complex is formed by adding an antibody to an antigen-containing sample, and then an antibody-binding protein-calciquestrin (CSQ) fusion protein is added to the antigen-antibody complex to form an antigen-antibody-antibody. forming a binding protein-CSQ complex; or (A2) binding an antibody and an antibody-binding protein-calciquestrin (CSQ) fusion protein to form an antibody-antibody-binding protein-CSQ complex, and then adding the antibody-antibody-binding protein-CSQ complex to a sample containing an antigen adding an antigen-antibody-antibody-binding protein-CSQ complex to form;
  • CSQ antibody-binding protein-calciquestrin
  • the antibody-binding protein is characterized in that it is a Z-domain, protein A, protein G or protein A/G.
  • FIG. 1 An exemplary schematic diagram of an immunoprecipitation method using an antibody binding protein and a calciquestrin fusion protein according to the present invention is shown in FIG. 1 .
  • the antibody-binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 4 to 6 or a Z-domain consisting of the amino acid sequence of any one of SEQ ID NOs: 4 to 6, SEQ ID NO: 7
  • Protein A comprising or consisting of the amino acid sequence of SEQ ID NO: 7
  • protein G comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 9 or comprising the amino acid sequence of SEQ ID NO: 9
  • It is characterized in that it is a protein A/G consisting of a sequence.
  • the method further comprises centrifuging the antigen-antibody-antibody binding protein-CSQ complex before step (C).
  • the method further comprises (D) performing Western blotting using an antigen-specific antibody.
  • the calcium concentration is 1 mM to 100 mM.
  • the sample is at least one selected from the group consisting of tissue, cells, whole blood, serum, plasma, tissue autopsy sample, cell culture supernatant, ruptured eukaryotic cells, and bacterial expression system. .
  • a Z-domain-calciquestrin fusion protein comprising a Z-domain and calciquestrin having the amino acid sequence shown in Table 1 below was designed.
  • Example 2 Turbidity by calcium of Z-CSQ1 and ZZ-CSQ1 fusion proteins
  • the turbidity of the Z-CSQ1 and ZZ-CSQ1 fusion proteins was measured at a concentration of each protein of 1 mg/ml. 20mM Tris-HCl (pH7.0) was used as a buffer for the fusion protein, and calcium was added to the fusion protein at concentrations of 0, 2, 4, 5, 6, 8, 10, 15 and 20 mM and mixed, The fusion proteins were reacted at 4° C. for 1 hr to react with calcium. Absorbance values for each concentration were measured in UV-Vis (350 nm) using Nano-drop (Thermo). As a result, it was confirmed that aggregates were formed from 2 mM calcium for both Z-CSQ1 and ZZ-CSQ1 fusion proteins ( FIG. 2 ).
  • Example 3 Measurement of size by calcium of Z-CSQ1 and ZZ-CSQ1 fusion proteins
  • the concentration of each protein was measured at 1 mg/ml. 20mM Tris-HCl (pH7.0) was used as a buffer for the fusion protein, and after adding and mixing 0 and 2mM calcium to the fusion protein, each fusion protein was reacted with calcium for 1 hr at 4°C. . Hydrodynamic size was measured using Zeta Sizer Nano Range (ELSZ-2000, Otsuka). As a result, it was confirmed that both the Z-CSQ1 and ZZ-CSQ1 fusion proteins formed aggregates with a size of 2,000 nm from a calcium concentration of 2 mM ( FIG. 3 ).
  • E. coli Escherichia coli
  • Trastuzumab Herceptin
  • ZZ-CSQ1 at a concentration of 3 mg/ml was added to the reaction solution and reacted at 4° C. for 30 minutes, and after calcium was added, the reaction was again performed at 4° C. for 30 minutes.
  • ProteinA-Agarose was reacted in the same manner.
  • the BSA antibody reaction solution was prepared by mixing Bovine Serum Albumin (BSA)-Antibody with the supernatant obtained by sonicating the Escherichia coli (E. coli) culture prepared in Example 5 and centrifugation.
  • BSA Bovine Serum Albumin
  • ZZ-CSQ2 fusion protein and ProteinA-Agarose were added, respectively, and reacted at 4°C for 30 minutes.
  • 5 mM calcium was added to the ZZ-CSQ2 fusion protein and reacted at 4° C. for 30 minutes.
  • Example 5 In order to confirm the immunoprecipitation of Z-CSQ1 fusion protein and ProteinA-Agarose, the culture medium of Escherichia coli (E. coli) prepared in Example 5 was sonicated and centrifuged in the supernatant. Bovine Serum Albumin (BSA) and BSA-antibody A reaction solution was prepared. Z-CSQ1 and ProteinA-Agarose were added and reacted at 4°C for 30 minutes, then calcium was added to Z-CSQ1 and reacted at 4°C for 30 minutes.
  • BSA Bovine Serum Albumin
  • BSA-antibody A reaction solution was prepared.
  • Z-CSQ1 and ProteinA-Agarose were added and reacted at 4°C for 30 minutes, then calcium was added to Z-CSQ1 and reacted at 4°C for 30 minutes.
  • Example 5 To confirm the immunoprecipitation of Trastuzumab between ZZ-CSQ1 fusion protein and ProteinA-Agarose, the culture medium of Escherichia coli (E. coli) prepared in Example 5 was sonicated and centrifuged. A reaction solution containing Trastuzumab and HER2 antigen. was prepared. ZZ-CSQ1 and ProteinA-Agarose were added and reacted at 4°C for 30 minutes, then calcium was added to ZZCSQ1 and reacted at 4°C for 30 minutes.
  • E. coli Escherichia coli
  • Example 5 In order to confirm the immunoprecipitation of ZZ-CSQ2 fusion protein and ProteinA-Agarose, the antibody reaction solution prepared in Example 5 was used. ZZ-CSQ2 and ProteinA-Agarose were added to the reaction solution containing Vascular Endothelial Growth Factor (VEGF) and Avastin, and after reacting at 4°C for 30 minutes, calcium was added to ZZ-CSQ2 and reacted at 4°C for 30 minutes.
  • VEGF Vascular Endothelial Growth Factor
  • the antibody reaction solution prepared in Example 5 was used to confirm the immunoprecipitation of the Z(5)-CSQ fusion protein and ProteinA-Agarose.
  • Z(5)-CSQ and ProteinA-Agarose were added to the reaction solution containing Bovine Serum Albumin (BSA) and BSA-antibody, and reacted at 4°C for 30 minutes. Then, calcium was added to Z(5)-CSQ and heated at 4°C. The reaction was carried out for 30 minutes.
  • BSA Bovine Serum Albumin
  • BSA-antibody Bovine Serum Albumin
  • Example 5 In order to confirm the immunoprecipitation of the CSQ-proteinA fusion protein and ProteinA-Agarose, the antibody reaction solution prepared in Example 5 was used. CSQ-proteinA and ProteinA-Agarose were added to a reaction solution containing Bovine Serum Albumin (BSA) and BSA-antibody, and calcium was added to CSQ-proteinA, which was reacted at 4°C for 30 minutes, and reacted at 4°C for 30 minutes. Each reaction solution is centrifuged at 4°C for 30 minutes to divide into a supernatant and a precipitate, and the precipitate is dissolved in 20 mM Tris-HCl (pH 7.0) buffer. 4X sample buffer was added, and after heat treatment at 80° C.
  • BSA Bovine Serum Albumin
  • the antibody reaction solution prepared in Example 5 was used to confirm the immunoprecipitation of the CSQ-proteinG fusion protein and ProteinA-Agarose.
  • CSQ-proteinG and ProteinA-Agarose were added to the reaction solution containing Bovine Serum Albumin (BSA) and BSA-antibody, and calcium was added to CSQ-proteinG, which was reacted at 4°C for 30 minutes, and reacted at 4°C for 30 minutes.
  • Each reaction solution was centrifuged at 4°C for 30 minutes to divide the supernatant and precipitate into a supernatant and a precipitate, and the precipitate was dissolved in 20 mM Tris-HCl (pH 7.0) buffer.

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Abstract

La présente invention concerne l'utilisation d'une protéine de liaison à un anticorps et d'une protéine de fusion de calséquestrine, et spécifiquement : une protéine de liaison à un anticorps et une protéine de fusion de calséquestrine pour l'immunoprécipitation ; un kit d'immunoprécipitation utilisant la protéine de liaison à l'anticorps et la protéine de fusion de calséquestrine ; et un procédé d'immunoprécipitation utilisant la protéine de liaison à l'anticorps et la protéine de fusion de calséquestrine.
PCT/KR2021/019070 2020-12-18 2021-12-15 Procédé d'immunoprécipitation utilisant une protéine de liaison à un anticorps et une protéine de fusion de calséquestrine WO2022131788A1 (fr)

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KR1020200177994A KR102612555B1 (ko) 2020-12-18 2020-12-18 항체결합단백질 및 칼시퀘스트린 융합단백질을 이용한 면역침강 방법

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US20100204455A1 (en) * 2007-07-27 2010-08-12 Pfizer Limited Antibody Purification Process By Precipitation
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KR20190026462A (ko) * 2017-09-05 2019-03-13 주식회사 바이오솔루션 온도에 따라 용해도가 달라지는 단백질을 포함하는 항체결합 침전체를 이용한 면역침강 방법

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EP3540438A1 (fr) 2012-08-17 2019-09-18 MorphoSys AG Anticorps propres à un complexe, fragments d'anticorps et leurs utilisations

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Publication number Priority date Publication date Assignee Title
KR20090113264A (ko) * 2007-01-22 2009-10-29 제넨테크, 인크. 항체의 고분자전해질 침전 및 정제
US20100204455A1 (en) * 2007-07-27 2010-08-12 Pfizer Limited Antibody Purification Process By Precipitation
US20130266930A1 (en) * 2010-12-15 2013-10-10 Cytosed, Inc. Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same
KR20170069452A (ko) * 2015-12-11 2017-06-21 한국세라믹기술원 Z-도메인 및 칼시퀘스트린 융합 단백질을 이용한 항체의 분리 및 정제 방법
KR20190026462A (ko) * 2017-09-05 2019-03-13 주식회사 바이오솔루션 온도에 따라 용해도가 달라지는 단백질을 포함하는 항체결합 침전체를 이용한 면역침강 방법

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