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     

Technological progresses in monoclonal antibody production systems

Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large‐scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on‐line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Mechanically induced orientation of adult rat cardiac myocytes in vitro

Summary A population of freshly isolated adult rat cardiac myocytes is spatially oriented using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 μm/min for 120 to 30 min, respectively, during the cell attachment period in serum-free medium induces a significant three-fold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. In contrast, adult myocytes plated onto a substratum undergoing continuous 10% stretch-relaxation cycling show no significant change in myocyte orientation or cytoskeletal organization. Orientation of rod-shaped myocytes is dependent on several factors other than the type of mechanical activity. These include: a) the speed of substratum movement; b) the final stretch amplitude; and c) the timing between initiation of substratum stretching and adhesion of myocytes to the substratum. Oriented adult rod shaped myocytes representing 65 to 70% of the total myocyte population in this model system can now be submitted to different patterns of repetitive mechanical stimulation for the study of stretch-induced alterations in cell growth and gene expression. This work was supported by grants AR36266, AR39998, and RR05818 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. J.-L. Samuel was a recipient from the Foundation pour la Recherche Médicale.     

A monoclonal antibody identifying a T-cell marker in the horse

A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23A precipitated a molecule of approximately 69 kDa from 125Iodine labelled horse lymphocytes.     

Sponge aggregation factor: in situ localization by fluorescent monoclonal antibody techniques

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction.     

Effects of peptone on hybridoma growth and monoclonal antibody formation

Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l^–1 range. It was found that 1–5 g l^–1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l^–1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l^–1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l^–1.     

In vitro quantification by image analysis of inotropic and chronotropic effects of drugs on cultures of cardiac myocytes

Quantitative image analysis is used to measure the inotropic and chronotropic effects of drugs on cultured heart cells maintained at 37°C on the stage of an inverted light microscope, and sequentially superfused with control and treatment media. The beating of the cardiac myocytes is evaluated by simultaneously selecting up to eight areas, including cell edges, from digitized video image. The sizes and positions of these areas are controlled by the operator. To analyze the motion of cell edges in each area, the computer measures the shift of the mass center of pixels' grey levels. Finally, a few parameters are calculated for the eight areas and displayed graphically. In order to assess treatment effects, appropriate statistical tests are performed on the data. Image analysis is an efficient screening test for evaluating the pharmacologic or toxic effects of a substance on isolated or cultured cardiac myocytes from various species.Abbreviations MEM Minimum Essential Medium     

Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody

Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

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%。     

A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.     

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.     

Real-time monitoring of protein secretion in mammalian cell fermentation: measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM)

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. (c) 1995 John Wiley & Sons, Inc.     

Uptake by rat liver of bovine growth hormone free or bound to a monoclonal antibody

Summary— In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh.     

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. (c) 1993 John Wiley & Sons, Inc.     

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.     

Influence of cell- and media-derived factors on the integrity of a human monoclonal antibody after secretion into serum-free cell culture supernatants

To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB-SF-21 AE) and four mammalian cell lines [CHO K1, BHK-21 (C13), C1271, P3-X63-Ag8.653], all of them commonly used for the production of recombinant proteins, and the human-human-mouse heterohybridoma cell line itself (H-CB-hahE), were adapted to serum-free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum-free media supplemented with 30 mug mL(-1) of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB-SF-21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well-known advantages in posttranslational modifications (e.g., formation of complex type N-glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus-infected insect cells. (c) 1995 John Wiley & Sons, Inc.