Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation

A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described illustrates many of the advantages of conventional batch fermentation processes but in addition has been shown to yield cell densities in excess of 1×10⁷ cells/ml with concomitant increase in product concentration.Abbreviations MAb Monoclonal Antibody - STR Stirred Tank Reactor - FBS Foetal Bovine Serum     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseudomonas aeruginosa as revealed by monoclonal antibodies and Western blotting

Abstract Monoclonal antibodies raised against single serotype components of a Pseudomonas aeruginosa vaccine have been shown to bind to the O antigen region of lipopolysaccharide (LPS). Outer membrane (OM) proteins, prepared by detergent treatment of envelope fractions and by EDTA/sonication treatment of whole cells, were separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred to nitrocellulose membrane and reacted with LPS-specific monoclonal antibodies. The patterns produced revealed that many of the protein bands were in fact protein-LPS complexes.     

Preparation and characterization of monoclonal antibodies to human hexokinase type I

1. Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I. None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis. 2. All seven monoclonals recognize the two different subtypes of human hexokinase I equally well. Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides. 3. Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.     

Antigenic components of human glioblastoma multiforme and rat C6 glioma using monoclonal antibodies

With whole U87MG cells used as antigenic stimulant, two clones 1A5G6 and 1D3A3 secreted monoclonal antibodies which gave intense staining in monolayer cultures of the cells as ascertained by indirect immunofluorescence. Antibodies from clone 1A5G6 stained both the cytoplasm and the processes, and that from clone 1D3A3 stained only the cytoplasm and not the processes. 1A5G6 elicited no cross-reactivity towards human fetal and adult brain and lungs, liver, kidney or spleen, mouse neuroblastoma and melanoma, rat C₆ glioma, neuroblastoma X glioma hybrid and normal rat kidney cells. It gave 58–60% cross reactivity with the human neuroblastoma and T-cell leukemia cells. The antigenic comPonent has been identified to be a membrane protein of molecular weight 25–30 kilodaltons by immunoblotting. Using C₆ glioma cells as antigenic stimulant 19 clones which were positive for C₆ glioma cells, but negative for rat liver cells as inferred by indirect immunofluorescence were selected. Antibodies secreted by all these gave positive reaction towards normal rat kidney and fetal rat kidney cells in culture. Distinct identity of these clones were ascertained by discernible staining patterns in indirect immunofluorescence on C₆ glioma cells.     

Select host cell proteins coelute with monoclonal antibodies in protein A chromatography

The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.     

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins

Summary Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.Abbreviations HIV Human Immunodeficiency Virus - SIV Simian Immunodeficiency Virus - HTLVI Human T cell Leukaemia Virus - AIDS Acquired Immune Deficiency Syndrome - mAb Monoclonal Antibody - ELISA Enzyme Linked Immunosorbent Assay - PBS Phosphate Buffered Saline     

Biotyping of pathogenic fungi by the killer system and with monoclonal antibodies

Biotyping of pathogenic yeasts and hyphomycetes based on their suceptibility to selected killer yeasts and their reactivity with monoclonal antibodies are described. Both methods were used to differentiate fungi isolated from patients providing valuable epidemiological information on mycotic infections. The functional biotyping obtained with the two systems and the conventional auxenographic biocoding approaches commercially available for opportunistic yeasts are comparatively evaluated. The potential for biotyping of industrial fungal isolates is also discussed.     

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

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Immunological approach to characterize proacrosin and various acrosin forms in boar and man by monoclonal antibodies

Three different monoclonal rat antibodies, Acr1, Acr2, and Acr3, have been established against boar proacrosin. They are shown by enzyme-linked immunosorbent and immunoblot assays to react with boar proacrosin and several different acrosin molecules derived therefrom during activation. The epitopes detected by the three antibodies are different from each other, one being highly sensitive to reduction and periodate treatment. The antibodies crossreact with various proacrosin and acrosin molecules derived from human sperm extract; they also show indirect immunofluorescent staining of the acrosomal region of ejaculated sperm from normal men but fail to react with round-headed spermatozoa.     

Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells

The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology. By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external. On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer. Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Linking radiosilver to monoclonal antibodies reduced by ascorbic acia

Radiosilver-111 and Radiogold-199 were proposed by us (1) as suitable isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111T _1/2=7.45 d and Au-199T _1/2=3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dialysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1∶1, 1∶2, 1∶5, 1∶10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver efflux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The comparative results with the two techniques are described.     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na⁺/glucose cotransporter.     

Preclinical studies of monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Eighty percent of bladder cancers present as superficial disease. Many are multifocal, and apparently successful treatment is frequently followed by recurrence. The use of monoclonal antibodies (MAbs) to target radiotherapy to these tumors offers great potential, especially since they can be administered directly into the bladder (intravesically) bypassing many of the side effects encountered to date with systemic MAb-based therapy. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. Tumor size can be monitored by X-ray analysis after administration of urograffin. Comparative studies of two murine MAbs, BLCA-8, IgG3, and C1-137, IgG1, against malignant human bladder cancer cells have been performed. Radio-immunoconjugates produced with¹²⁵Iodine (¹²⁵I) have been used for biodistribution studies following administration directly into rat bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intravesical administration of radio-immunoconjugates to tumor-bearing nude rats indicate better tumor uptake of C1-137 than BLCA-8. Further studies to test two-step intravesical administration of biotinylated MAb followed by radioiodinated streptavidin are in progress. Our studies indicate that the C1-137 MAb may have considerable potential for intravesical radioimmunotherapy of patients with superficial bladder tumors.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

The peptidase activity of human serum butyrylcholinesterase: Studies using monoclonal antibodies and characterization of the peptidase

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co²⁺, Mn²⁺, and Zn²⁺ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co²⁺, Mn²⁺, and Zn²⁺ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited -chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.