High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016     

Preparation of a monoclonal antibody to a melanoma growth-stimulatory activity released into serum-free culture medium by Hs0294 malignant melanoma cells

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.     

表达抗-CD25单克隆抗体的GS-NS0骨髓瘤细胞无血清培养及代谢特性

抗-CD25单克隆抗体作为免疫抑制剂拥有广阔的市场前景和巨大的经济价值。本实验以表达抗?CD25单克隆抗体的GS-NS0细胞为研究对象,开发了支持其大规模培养和抗体表达的无血清低蛋白培养基,批培养最大活细胞密度和最大抗体浓度分别达3×106cells/mL和300mg/L以上,比商业无血清培养基(Excell 620+0.2% primatone)分别提高了100%和46%。通过批培养实验,研究了细胞的生长、葡萄糖和氨基酸代谢、以及产物表达特点,并揭示了批培养过程中初始葡萄糖浓度对GS-NS0细胞生长与代谢的影响规律。为优化GS-NS0细胞培养过程和抗CD25单抗成功迈向产业化提供了重要的科学依据。     

Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

During a small‐scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small‐scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:562–570, 2014     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.     

Localization and partial characterization of a rat ovarian granulose cell protein with a monoclonal antibody

Summry— Hybridoma cell lines were obtained from mouse splenocytes sensitized to granulosa cells collected from rat ovaries after gonadotropin stimulation. A monoclonal antibody (5G5) was obtained which reacted with granulosa cells and showed a positive reaction with serum-free conditioned medium containing granulosa cell secreted proteins. Immoblotting of the conditioned medium and light- and electron-microscopic immunocytochemistry of rat ovary show that mAb 5G5 is directed against a 59-kDa protein which is located on the plasma membrane of granulosa cells. Furthermore, the immunoreactivity of the granulosa cells depends both on the degree of follicle development and on the position of the granulosa cells within the follicles. Strong immunoreactivity was observed in the innermost granulosa cell layers, close to the oocyte and the antral cavity. The results obtained show that mAb 5G5 is a useful marker of a 59-kDa granulosa cell protein which might be of importance for the follicle and the occyte maturation.     

CHO细胞表达抗血管内皮生长因子单克隆抗体工艺优化

抗血管内皮生长因子单克隆抗体(VEGF-MA)能够抑制肿瘤生长,具有良好的市场前景。本研究利用一株重组中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)细胞株表达VEGF-MA。首先对培养基种类进行优化,筛选最优的基础培养基、补料培养基和外源添加物。研究结果表明:最有利于重组CHO细胞株表达VEGF-MA的基础培养基、补料培养基和外源添加物分别为ActiCHO P Powder CD、CD Efficient Feed C AG和Sheff-CHO Plus PF ACF。利用这一培养基配比在摇瓶和3 L发酵罐中培养重组CHO细胞,VEGF-MA产量均可达到3.20 g/L。在3 L发酵罐中进一步优化培养条件,结果表明:最优的接种密度、pH、溶解氧浓度、前期培养温度和后期培养温度分别为1.0×10^6个/mL、7.10、40%、36.5℃和34℃,此时的VEGF-MA产量能够达到4.10 g/L。VEGF-MA质量指标均处于标准范围内:电荷异质性、糖基化水平和蛋白纯度分别为26.1%、59.1%和95.1%。     

Modulation of high mannose levels in N-linked glycosylation through cell culture process conditions to increase antibody-dependent cell-mediated cytotoxicity activity for an antibody biosimilar

The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N-glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity. In this study, we describe efforts to modulate the high-mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed-batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N-glycosylation were investigated, including osmolality, ammonium chloride (NH₄Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale-up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Role of iron and sodium citrate in animal protein-free CHO cell culture medium on cell growth and monoclonal antibody production

Chemically defined iron compounds were investigated for the development of animal protein-free cell culture media to support growth of CHO cells and production of monoclonal antibodies (mAb). Using a multivessel approach of 96-well plates, shake flasks, and bioreactors, we identified iron and its chemical partner citrate as critical components for maintenance of continuous cell growth and mAb production. The optimized iron concentration range was determined to be 0.1-0.5 mM and that for citrate 0.125-1 mM. This complete formulation is able to maintain cell growth to similar levels as those supplemented with iron compounds alone; however, mAb productivity was enhanced by 30-40% when citrate was present. The addition of sodium citrate (SC) did not affect product quality as determined by size exclusion chromatography, ion exchange chromatography, reversed phase and normal phase-HPLC. No significant changes in glucose and lactate profiles, amino acid utilization, or mAb heavy and light chain expression ratios were observed. Cellular ATP level was ～30% higher when SC was included suggesting that SC may have a role in enhancing cellular energy content. When cell lysates were analyzed by LC-MS to assess the overall cellular protein profile, we identified that in the SC-containing sample, proteins involved in ribosome formation and protein folding were upregulated, and those functions in protein degradation were downregulated. Taken together, this data demonstrated that iron and citrate combination significantly enhanced mAb production without altering product quality and suggested these compounds had a role in upregulating the protein synthetic machinery to promote protein production.     

Host cell protein dynamics in the supernatant of a mAb producing CHO cell line

The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.     

A comparative study of the bispecific monoclonal antibody,blinatumomab expression in CHO cells and E. coli

Abstract

The “bispecifics” market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6?×?His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS–PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55?kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.     

Preparation and characterization of a human aurora-A kinase monoclonal antibody

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an Elisa (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.