Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes.

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.

[1]  B. Mattiasson,et al.  Conjugates of monoclonal antibodies with polyelectrolyte complexes-- an attempt to make an artificial chaperone. , 1998, Biochimica et biophysica acta.

[2]  B. Mattiasson,et al.  Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes , 1998, Bioseparation.

[3]  B. Mattiasson,et al.  Polycomplexes – potential for bioseparation , 1998, Bioseparation.

[4]  M. Delepierre,et al.  Does a peptide bound to a monoclonal antibody always adopt a unique conformation? , 1998, Research in immunology.

[5]  Y. Osawa,et al.  Preparation and characterization of the Fab and F(ab′)2 fragments of an aromatase activity-suppressing monoclonal antibody , 1997, Steroids.

[6]  V. Muronetz,et al.  A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate. , 1997, Archives of biochemistry and biophysics.

[7]  T. Kouki,et al.  Separation method of IgG fragments using protein L. , 1997, Immunological investigations.

[8]  J. Frank,et al.  Three-dimensional reconstruction of Androctonus australis hemocyanin labeled with a monoclonal Fab fragment. , 1995, Journal of structural biology.

[9]  T. van der Poll,et al.  Complete Inhibition of Endotoxin-Induced Coagulation Activation in Chimpanzees with a Monoclonal Fab Fragment against Factor VII/VIIa , 1995, Thrombosis and Haemostasis.

[10]  Y. Li,et al.  Preparation, characterization and crystallization of an antibody Fab fragment that recognizes RNA. Crystal structures of native Fab and three Fab-mononucleotide complexes. , 1995, Journal of molecular biology.

[11]  J. Rousseau,et al.  Evaluation of a 99mTc-antimyosin kit for myocardial infarct imaging. , 1994, Journal of nuclear biology and medicine.

[12]  V. Kabanov.  Physicochemical basis and the prospects of using soluble interpolyelectrolyte complex (review) , 1994 .

[13]  T. Teeri,et al.  Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain. , 1993, Gene.

[14]  S. Nakai,et al.  Production and purification of Fab' fragments from chicken egg yolk immunoglobulin Y (IgY). , 1993, Journal of immunological methods.

[15]  M. Bodmer,et al.  Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment. , 1992, The Biochemical journal.

[16]  H. Schwarz,et al.  High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold. , 1991, Journal of structural biology.

[17]  R. W. Baldwin,et al.  Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments. , 1989, Journal of immunological methods.

[18]  S. Boguslawski,et al.  Improved procedure for preparation of F(ab')2 fragments of mouse IgGs by papain digestion. , 1989, Journal of immunological methods.

[19]  S. Pond,et al.  Large scale production and purification of paraquat and desipramine monoclonal antibodies and their Fab fragments. , 1988, International journal of immunopharmacology.

[20]  B. Friguet,et al.  Epitope localization in antigen-monoclonal-antibody complexes by small-angle X-ray scattering. An approach to domain organization in the beta 2 subunit of Escherichia coli tryptophan synthase. , 1987, European journal of biochemistry.

[21]  J. Bidlack,et al.  Preparation of Fab fragments from a mouse monoclonal IgM. , 1986, Journal of immunological methods.

[22]  H. Bazin,et al.  Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. , 1983, Journal of immunological methods.

[23]  A. Margolin,et al.  Preparation and properties of penicillin amidase immobilized in polyelectrolyte complexes. , 1981, Biochimica et biophysica acta.

[24]  Y. Klemes,et al.  Cross-linking preserves conformational changes induced in penicillinase by its substrates. , 1980, The Biochemical journal.

[25]  S. Lykkegård.  Purification and characterization of rabbit anti-mouse renin specific Fab fragments. , 1979, Acta pathologica et microbiologica Scandinavica. Section C, Immunology.

[26]  T. Cherednikova,et al.  [Enzymatic splitting of antibodies bound to immobilized antigen as a means of Fab fragments preparation]. , 1976, Biokhimiia.

[27]  J. H. Park,et al.  Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography. , 1975, Analytical biochemistry.

[28]  L. Sluyterman,et al.  The effect of salts upon the pH dependence of the activity of papain and succinyl-papain. , 1972, Biochimica et biophysica acta.

[29]  A. Fensleau.  Structure-function studies on glyceraldehyde 3-phosphate dehydrogenase. IV. Subunit interactions of the rabbit muscle and yeast enzymes. , 1972, The Journal of biological chemistry.

[30]  M. Jonsson.  Isoelectric spectra of native and base denatured crystallized swine pepsin. , 1972, Acta chemica Scandinavica.

[31]  Porter Rr,et al.  The interchain disulfide bonds of a human pathological immunoglobulin. , 1967 .

[32]  R. Porter.  The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. , 1959, The Biochemical journal.