Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Affinity chromatography and co-chromatography of bispecific monoclonal antibody immunoconjugates

Bispecific monoclonal antibodies (bsMAb) are unique macromolecules functioning as cross-linkers with two different predetermined binding specificities. A wide range of potential applications employing these probes can be envisioned in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid-hybridomas is the production of parental monospecific antibodies along with bsMAbs. Hence, the purification of desired bsMAb free from both parental mAbs and other possible promiscuous combinations is essential. Purification of antibodies is the single greatest obstacle in obtaining an immunoprobe with high specific activity. This review describes the affinity purification and affinity co-purification techniques for the separation of bsMAb as a pre-formed immune complex or as a pure species. The use of immobilized ligands is the basis of affinity chromatography. Affinity chromatography can be classified into three different categories depending on the properties of the immobilized ligand. The ligand-specific affinity chromatography is based on the extremely specific immobilized ligand, directed towards the protein or antibody of interest. Using a dual, sequential affinity chromatography, bsMAb can be purified from a mixture of bispecific and monospecific monoclonal antibodies with a ligand specific for each antibody. Thiophilic adsorption is a group-specific affinity method that can be successfully used to separate monospecific forms from bispecific species by salt gradient elution. Affinity co-chromatography offers a convenient one-step method for purification of bulk amounts of immunoconjugates for diagnostic applications by exploiting several dye-ligands known to bind certain enzymes. The same method could be potentially used for quality control and quality assurance purposes in industrial biotechnology.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.     

Monoclonal Antibody Developed against a Hemolysin of Bacillus thuringiensis

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt-hemolysin but also to a hemolysin (Bc-hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt- and Bc-hemolysins by immunoaffinity column chromatography. The partial N-terminal amino acid sequence of the purified hemolysins was determined to be Ile-Glu-Gln-Thr.     

Monoclonal antibodies to plasma high density lipoprotein (HDL) of eel (Anguilla japonica)

Six week-old female mice (Balb/c) injected intraperitonealy with 50 μg of eel high density lipoprotein (HDL) emulsified with equal volume of adjuvant three times every two weeks. Three weeks after the third injection, hyperimmunized mice were boosted by injection of 100 μg of HDL. After 5 days, the best responding mouse to injected HDL was sacrificed, and spleen cells were fused with mouse myeloma cells (Sp2/O–Ag14), and hybridomas were cultured in a selection medium. Monoclonal antibodies specific to apolipoprotein A-I or A-II (apoA-I or apoA-II) of HDL were obtained by cloning and recloning the hybridomas. Eighteen monoclonal antibodies specific to apoA-I and/or apoApII were isolated. Antibodies in the culture medium were purified by a HiTrap Protein G or an eel-HDL column. These purified antibodies belong to the subclass IgG1. The monoclonal antibodies specific to eel apoA-I and apoA-II secreted by clone 10D12 and 2G3, respectively, interact with serum proteins of some fish species such as red-sea bream and carp. The anti-eel apoA-I antibody of 10D12 did not bind to serum proteins of rat, rabbit, and chicken, while the anti-eel apoA-II of 2G3 antibody did.     

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and⁶⁷Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%–40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.     

Establishment of a mouse hybrid-hybridoma secreting bispecific antibodies to bovine lactoferrin and horseradish peroxidase

Summary A mouse hybridoma HS@03A secreting anti-horseradish peroxidase (HRPO) monoclonal antibodies (IgG1) was established. A HAT sensitive clone of HS@03A was obtained by culturing the hybridoma cells in a 6-thioguanine supplemented medium. The resulting clone 03AR10-2 was fused with a 5-bromo-2-deoxyuridine resistant (HAT sensitive) clone of a mouse hybridoma HB8852 secreting anti-bovine lactoferrin (bLF) antibodies. Hybrid-hybridomas secreting bispecific antibodies were selected and a hybrid-hybridoma clone HH1-4-3 was established. The bispecific antibodies secreted by the hybrid-hybridoma HH1-4-3 were found to be useful for the analysis of bLF by competitive ELISA.     

抗iLRP单克隆抗体的制备和鉴定

为了研究抗iLRP (Immature laminin receptor protein)单克隆抗体在免疫治疗中的应用,本研究采用重组iLRP对6~8周龄BALB/c雌性小鼠进行免疫,取免疫后小鼠脾细胞与骨髓瘤细胞系SP2/0进行细胞融合,在HAT (Hypoxanthine aminopterin thymidine)半固体培养基上挑选分离明显的细胞克隆于96孔板培养后,取上清液通过ELISA法进行克隆筛选,初次筛选得到23株阳性克隆,复筛后得到13株稳定分泌抗体的细胞株。抗体类别鉴定显示,其中9株为IgM,3株为Ig G1,1株为IgG2a,轻链类型都为κ型。其中1株IgG1和IgG2a用于制备小鼠腹水,经Protein-G柱纯化,SDS-PAGE检测抗体纯度,Western blotting验证抗体特异性。结果表明,抗体纯度均大于90%,并与i LRP有明显的特异性结合。此结果为后续的深入研究提供了一套完整有效的抗iLRP单克隆抗体生产和鉴定的程序,对以iLRP为治疗靶点的单克隆药物研究以及最近兴起的CAR-T (Chimeric antigen receptor modified T cell)免疫治疗具有积极的意义。     

A human hybrid hybridoma

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. We have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. We fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1 kappa antibody directed against tetanus toxoid and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1 lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassays. Our results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies.     

The demonstration of proliferating cells by using monoclonal antibodies to 5-bromo-2'-deoxyuridine in the peroxidase-antiperoxidase method

Monoclonal antibodies (MAb) to the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd) and to horseradish peroxidase (HRPO) have been produced and characterized. On the basis of Mab to HRPO, complexes of the antibodies and HRPO (PAP-complexes) for immunochemical investigations are prepared. The possibility to identify proliferating cells in cultures of mouse myeloma Sp2/0 and mouse fibroblasts NIH 3T3 using Mab to BrdURd by the PAP-method is shown. The conditions of performing the analysis were optimized. The effect of various techniques ot cell fixation and cell DNA denaturation on cell morphology and on specific staining of nuclei of BrdUrd-containing cells in investigated.     

Utilization of leucine methyl ester for the generation of hybridomas producing monoclonal antibodies specific to tumor-associated antigens

In an attempt to obtain monoclonal antibodies specific to tumor-associated antigens. C3H/He mice were immunized with syngeneic MM2 tumor cells, and the primed spleen cells were fused with P3-X63-Ag8.653 myeloma cells. The outgrowth of hybridomas, however, was extremely low and monoclonal antibodies were not obtained. The reason for the low hybridoma growth was studied. It was found that MM2 cells used as the immunogen, the fusion partner myeloma cells and the resulting hybridomas shared at least one tumor-associated antigen, namely Q5 antigen. Because of this common antigen, cytotoxic cells, presumably cytotoxic T lymphocytes, which were lytic to the hybridomas, were induced during the culture for generation of the hybridomas. Removal of lysosome-rich cells, including cytotoxic T lymphocytes, from the primed spleen cells before the fusion by treatment with leucine methyl ester, a lysosomotropic agent, drastically improved the outgrowth of hybridomas. By this method, seven stable hybridoma clones producing monoclonal antibodies specific to tumor-associated antigens were obtained. Two of the seven clones were found to secrete monoclonal IgM species, which reacted with the extra-cellular region of the Q5 antigen. This procedure will be an option when production of monoclonal antibodies specific to cell-surface antigens is intended and outgrowth of hybridomas is unexpectedly low.     

Production, characterization and applications of mouse anti-grass carp (Ctenopharyngodon idellus) growth hormone monoclonal antibodies

Mouse anti-grass carp growth hormone (gcGH) monoclonal antibody (MAb) secretors were produced by PEG-mediated fusion of NS-1 myeloma cells and splenic B-lymphocytes of gcGH hyper-immunized mice. Positive secretors were screened by direct ELISA and cloned by limiting dilution. Three positive secretors, 21D3, 22G5 and 23B3, were obtained in a single fusion trial. Anti-gcGH MAbs were produced by growing hybridomas in the peritoneal cavity of pristane-primed mouse. The three MAbs were isotyped to be IgG2a, IgG2b and IgM, respectively. IgG MAbs were purified from ascitic fluid by Hitrap protein G column and IgM MAb was purified by gel filtration chromatography. The purified MAbs were highly specific and had moderate binding affinity. The MAbs were successfully used for the purification of native gcGH from mature grass carp pituitary extract by one-step immunoaffinity chromatography, for the quantification of gcGH by competitive sandwich ELISA, and for the probing of somatotropes in grass carp pituitary by immunohistochemistry.     

Immunohistochemical localization of polysialic acid in tissue sections: differential binding to polynucleotides and DNA of a murine IgG and a human IgM monoclonal antibody.

For immunolocalization of alpha(2-8)-linked polysialic acid, which forms part of the neural cell adhesion molecule (N-CAM), two monoclonal antibodies, MAb735 and IgMNOV, were employed. Both antibodies have previously been shown to bind the extremely low immunogenic capsular polysaccharide of group B meningococci, which also consists of alpha(2-8) polysialic acid, but not to other, even closely related forms of polysialic acid. Despite the identical polysaccharide specificity of these two MAb, we observed marked differences of the staining pattern in tissue sections. We showed that these differences in immunostaining were due to the crossreactivity of IgMNOV with polynucleotides and DNA. MAb735, however, was shown to react exclusively with alpha(2-8) polysialic acid. Moreover, the specificity of MAb735 proved to be unique among eleven other MAb directed against various bacterial polysaccharides, as it was the only one unreactive with polynucleotides. Thus, MAb735, the only IgG type mouse monoclonal antibody to polysialic acid thus far reported, can be considered a specific probe for the unambiguous detection of alpha(2-8) polysialic acid in tissue sections, and should therefore help to further elucidate the role of polysialic acid in developmental processes.     

Media for hybridoma growth and monoclonal antibody production

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC–HILIC) column coupled with electrospray ionization mass spectrometry (ESI–MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC–HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC–HILIC column and ESI–MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC–HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.     

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.     

A synthetic ligand for IgA affinity purification

We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support.