Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Isolation of recombinant human antithrombin isoforms by Cellufine Sulfate affinity chromatography.

Human antithrombin (hAT) is a major serine protease inhibitor that regulates blood coagulation in human plasma, and it has been applied for the treatment of antithrombin (AT) deficiency and disseminated intravascular coagulation (DIC). In the past, hAT for therapeutic use has been obtained from human plasma; however, hAT can now be sourced from transgenic animals and Chinese hamster ovary (CHO) cells by recombinant technology. The dominant form of hAT in plasma is the α form, which is glycosylated with four oligosaccharides and sialylated at its terminals. However, it would be preferable to remove the poorly sialylated α form of hAT, the minor ß form with unoccupied glycosylation sites, and the inactive forms. Cellufine Sulfate, a heparin-mimic affinity resin made of cellulose and modified with sulfate groups, has an affinity for heparin-binding proteins and can be used in cation exchange chromatography. Based on these properties, the α and ß forms of hAT could be separated and purified depending on the rate of sialylation. Consequently, Cellufine Sulfate was used to enrich the highly sialylated α form with a considerably higher step yield than that obtained using conventional heparin-immobilized resins. Furthermore, subsequent hydrophobic interaction chromatography could eliminate the inactive forms. These results suggested that Cellufine Sulfate was more effective than heparin-immobilized resins in purifying the highly sialylated α form of hAT for therapeutic applications.

The therapeutic potential of a novel PSMA antibody and its IL-2 conjugate in prostate cancer.

Prostate-specific membrane antigen (PSMA) is an attractive target for treatment of prostate cancer. Using the PSMA-recognizing mouse monoclonal antibody 2C9 obtained in our previous study, the biological activities of PSMA antibody were evaluated. Mouse-human chimeric IgG1 of 2C9 (KM2777) showed antibody-dependent cellular cytotoxicity activity against PSMA-expressing prostate cancer cells in the presence of human peripheral blood mononuclear cells (PBMCs). To increase lymphocyte-mediated cytotoxicity of KM2777, C-terminus interleukin-2 (IL-2)-fused KM2777 (KM2812) was constructed. KM2812 retained binding activity to PSMA and exhibited growth-stimulating activity equivalent to IL-2 on the IL-2-dependent T-cell line CTLL-2. Moreover, KM2812 exhibited enhanced cytotoxic activity against PSMA-expressing prostate cancer cells in the presence of PBMCs compared with KM2777. In a xenograft tumor model using PSMA-expressing prostate cancer cells, KM2812 exhibited marked antitumor activity, accompanied by complete regression of tumor in some of the KM2812-treated mice. These results suggest that KM2812 has a therapeutic potential for prostate cancer by stimulating lymphocyte-mediated antitumor cytotoxicity.

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines.

L-alanyl-L-glutamine (AlaGln) is dipeptide that has better solubility and stability than Glutamine (Gln). In this study, we evaluated the utility of this dipeptide during culture of POTELLIGENT™ Chinese hamster ovary (CHO) cells expressing anti-CD20 chimeric antibody. Although AlaGln in the culture medium lowered the specific growth rate, the MAb titer was maximized when Gln was completely replaced by AlaGln in both the basal and feed media. Moreover, AlaGln augmented production of antibody not only at flask scale but also at spinner scale, although the extent of this effect was dependent on the cell clone. To explore the mechanism responsible for the effect of AlaGln on cell growth, we measured apoptosis in the early phase of cell culture on days 8, 9, and 10. The apoptotic ratio was reduced in medium containing AlaGln. Ammonia was generated in medium containing Gln when it was maintained at 37 °C, which impeded the growth and productivity of the cells. In contrast, AlaGln produced less ammonia under these conditions, which may have been one of the properties associated with its beneficial effects. We conclude that certain dipeptides can serve as superior alternative sources of amino acids in cell culture and antibody production.

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity.

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.

A novel liposome surface modification agent that prolongs blood circulation and retains surface ligand reactivity.

Liposomes are recognized as potentially useful drug carriers but many problems preclude practical medical application. Liposomes bind with serum proteins (opsonization) and are captured by the reticuloendothelial system cells in the liver and spleen, which limits their ability to deliver drugs to other target sites. Modification of lipids with flexible, hydrophilic polymers such as poly(ethylene glycol) (PEG) to yield sterically stabilized liposomes is one approach to improve liposome blood circulation and tissue distribution properties. In this study, we examined liposomes prepared using lipids modified with a new branched oligoglycerol (BGL) moiety for steric stabilization. This novel BGL comprised 14 glycerol units (termed BGL014) connected with flexible ether linkages, resulting in a branched cascade-like structure that is highly expanded in aqueous solution. BGL014 was coupled to 1,2-distearoylphosphatidylethanolamine to yield BGL014-modified lipids. Incorporation of BGL014 into liposomes (BGL014L) resulted in long blood circulation times, despite a much thinner fixed aqueous layer thickness compared to PEG formulations. BGL014 produced a liposome surface coating that appears to function through steric inhibition of non-specific protein binding without strong interference of specific protein-binding reactions. Liposome structure and functionality was maintained following BGL014-modification, as the incorporation ratio of drug remained high. These results suggest that the BGL014 modification of liposomes is a promising approach to produce stable and long circulating drug carriers capable of selective binding to specific proteins.

Preparation and properties of branched oligoglycerol modifiers for stabilization of liposomes.

We examined the effect on drug delivery of liposomes with surfaces that were modified with branched oligoglycerols (BGLs) and explored possible formulation advantages to increase drug exposure. BGL012 is a branched oligoglycerol derivative with a cascade-like structure of 12 glycerol units, characterized as a widely spread structure in aqueous solution. We prepared BGL-phospholipid derivatives (BGL-PEs), including BGL012, by coupling 1,2-distearoylphosphatidylethanolamine to BGLs. BGL012-PE modification of the liposomes (BGL012L) achieved a long circulation time after intravenous injection in rats. The circulation lifetime of BGL012L was almost the same as that of polyethylene glycol (PEG)-modified liposomes. The surface of BGL012L induced the formation of a fixed aqueous layer and reduced protein adsorption on the liposome surface, without strong interference with the binding reaction on the liposome. Thus, the newly synthesized branched oligoglycerol derivatives are considered to have useful hydrophilic and physical properties for modifying the liposome surface to increase drug exposure.

A chromatographic approach for elevating the antibody-dependent cellular cytotoxicity of antibody composites.

Recent studies have shown that antibodies with low fucose content in their oligosaccharides exhibit highly potent antibody-dependent cellular cytotoxicity (ADCC). However, composites of therapeutic antibodies produced by conventional production systems using cell lines such as Chinese hamster ovary (CHO) and SP2/0 do not necessarily contain sufficient amounts of non-fucosylated antibody species. In this study, we combined two lectin-affinity chromatography techniques, Concanavalin A and Lens culinaris agglutinin, to enrich the non-fucosylated species from therapeutic material using the anti-Her2/neu model antibody. Oligosaccharide analysis by matrix-assisted laser desorption/ionization-time of flight MS following peptide-N-glycosidase F digestion suggested that non-fucosylated antibody could be enriched in the purified fraction with efficient removal of high-mannose species. The ADCC activity of the purified fraction was about 100-fold higher than that of the initial material. The chromatographic strategy presented here can be a useful tool to elevate ADCC activity of antibody materials without concentrating high-mannose oligosaccharides.

Modification of protein with BGL06, a novel branched oligoglycerol derivative.

A branched oligoglycerol derivative, BGL06, with a cascade-like structure of glycerol units was used as a novel reagent for protein modification. Modification reaction with a recombinant human granulocyte-colony stimulating factor derivative, ND28, was carried out successfully in aqueous conditions to obtain a coupled form, BGL06-ND28. Characterization of the modified ND28 suggests that two types of products were obtained by controlling the reaction; one was H(BGL06)-ND28, a highly modified version coupled with 4.34 molecules of BGL06 units on average, and the other was L(BGL06)-ND28, a moderately modified version coupled with 2.58 molecules of BGL06 units on average, respectively. The properties of these products were compared to the known polyethylene glycol (PEG)-modified ND28. In the cell proliferation assays, unlike PEGylation, modification with BGL06 did not produce a significant loss of biological activity even when the modification extent was elevated. Under such conditions, 76.0% of the activity was in fact maintained for H(BGL06)-ND28, while PEGylated ND28 retained only 24.6% of biological activity in vitro even though the extent of modification was smaller. In addition, H(BGL06)-ND28 showed comparable thermostability to a 20 kDa PEG-modified counterpart. Therefore, the BGL06 derivative will be a useful alternative as a protein modification reagent where PEGylation is not effective.

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide.

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.