Specific Antibody Production Rate Research Articles

Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Characteristics of Viral Shedding in Respiratory Samples and Specific Antibodies Production in 564 COVID-19 Patients.

SummaryPositive nucleic acid (NA) results have been found in recovered and discharged COVID-19 patients, but the proportion is unclear. This study was designed to analyze the recurrent positive rate of NA results after consecutively negative results, and the relationship between the specific antibody production and positive NA rate. According to Strengthening the Reporting of Observational Studies in Epidemiology guidelines, data of inpatients in Sino-French New City Branch of Tongji Hospital between Jan. 28 and Mar. 6, 2020 were collected. A total of 564 COVID-19 patients over 14 years old who received the examinations of NA and antibodies against SARS-CoV-2 were included. Days of viral shedding and specific antibodies were recorded and assessed. Among NA tests in respiratory samples (throat swabs, nasopharyngeal swabs, sputum and flushing fluid in alveoli), the patients with all-negative NA results accounted for 17.20%, those with single-positive results for 46.63%, and those with multiple-positive results for 36.17% respectively. Besides, the recurrent positive NA results after consecutively negative results appeared in 66 patients (11.70%). For multiple-positive patients, median viral shedding duration was 20 days (range: 1 to 57 days). Of the 205 patients who received 2 or more antibody tests, 141 (68.78%) had decreased IgG and IgM concentrations. IgM decreased to normal range in 24 patients, with a median of 44 days from symptom onset. Viral shedding duration was not significantly correlated with gender, age, disease severity, changes in pulmonary imaging, and antibody concentration. It is concluded that antibody level and antibody change had no significant correlation with the positive rate of NA tests and the conversion rate after continuous negative NA tests. In order to reduce the recurrent positive proportion after discharge, 3 or more consecutive negative NA test results with test interval more than 24 h every time are suggested for the discharge or release from quarantine.

Comparative phenotypic analysis of CHO clones and culture media for lactate shift

We explored the effects of media and clonal variation on the lactate shift which can be considered as one of the desirable features in CHO cell culture. Various culture profiles with the specific growth and antibody production rates under three different media conditions in two CHO producing clones were evaluated by resorting to multivariate statistical analysis. In most cases, glutamine depletion coincided with lactate consumption, suggesting that glutaminolysis rather than glycolysis was the preferred pathway for the pyruvate supply toward lactate production. With respect to the lactate shift, high performing medium showed higher glutamate uptake, higher aspartate secretion and lower serine uptake compared to other media conditions. In addition, clone itself exhibited the desired lactate consumption more consistently accompanying with distinguishing phenotype. The clone exhibiting lactate shift produced lesser lactate in exponential phase but two-fold higher non-toxic alanine, thus leading to better culture environment. Thus, we understand the balanced selection of clone and media composition enables cells to utilize the metabolic pathways for the desired lactate shift.

Development of anti-hepatitis B surface (HBs) antibodies after HBs antigen loss in HIV-hepatitis B virus co-infected patients

BackgroundHepatitis B surface antigen (HBsAg)-seroconversion, or loss of HBsAg and acquisition of anti-hepatitis B surface (HBs) antibodies, defines functional cure of chronic hepatitis B virus (HBV) infection. After HBsAg-loss, little is known regarding the development of anti-HBs antibodies and even less so in individuals co-infected with HIV. ObjectivesTo determine anti-HBs antibody kinetics after HBsAg-loss and explore determinants of HBsAg-seroconversion in HIV-HBV co-infected patients. Study designPatients enrolled in the French HIV-HBV cohort were included if they had >1 study visit after HBsAg-loss. Individual patient kinetics of anti-HBs antibody levels were modeled over time using mixed-effect non-linear regression, whereby maximum specific growth rate and maximal level of antibody production were estimated from a Gompertz growth equation. ResultsFourteen (4.6%) of 308 co-infected patients followed in the cohort exhibited HBsAg-loss, all of whom were undergoing antiretroviral therapy. Nine (64.3%) of these patients achieved HBsAg-seroconversion during a median 3.0 years (IQR=1.1–5.1) after HBsAg-loss. Across individuals with HBsAg-seroconversion, the fastest rates of antibody growth ranged between 0.57–1.93year−1 (population maximum growth rate=1.02) and antibody production plateaued between 2.09–3.66 log10 mIU/mL at the end of follow-up (population maximal antibody levels=2.66). Patients with HBsAg-seroconversion had substantial decreases in HBV DNA viral loads (P=0.03) and proportion with elevated ALT levels (P=0.02) and HBeAg-positive serology (P=0.08). No such differences were observed in those without HBsAg-seroconversion. ConclusionsMost co-infected patients with HBsAg-seroconversion produced and maintained stable antibody levels, yet kinetics of anti-HBs production were much slower compared to those observed post-vaccination or after clearance of acute HBV-infection.

Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells

Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production.

Co-culture of the 55-6 B cell hybridoma with the EL-4 thymoma cell. Effect on cell growth and monoclonal antibody production

The cell growth and monoclonal antibody production of the 55-6 hybridoma cell co-cultured with the murine thymoma cell line EL-4 at different initial 55-6:EL-4 ratios were investigated. Both populations were seeded in co-culture without previous stimulation and therefore with low constitutive CD40 and CD40 ligand (CD154) expression levels, and in the absence of exogenous co-stimuli. Viable cell density and growth rate data seem to suggest a competition for nutrients, which is detrimental for both cells in terms of biomass production and also of growth rate for 55-6. Final concentrations of antibody and specific antibody production rates were affected by the initial 55-6:EL-4 ratio. The 4:1 ratio yielded the highest IgG2a concentration, whereas the highest specific antibody production rate was obtained at the 2:1 ratio. Changes mainly in CD154 and also in CD40 expression in co-cultures could suggest cross-talk between both populations. In conclusion, different types of interactions are probably present in this co-culture system: competition for nutrients, cognate interaction and/or autocrine or paracrine interactions that influence the proliferation of both cells and the hybridoma antibody secretion. We are hereby presenting a pre-scale-up process that could speed up the optimization of large-scale monoclonal antibodies production in bioreactors by emulating the in vivo cell-cell interaction between B and T cells without previous stimulation or the addition of co-stimulatory molecules.

Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.

Application of improved top-down approach in maximizing CHO cell mass and productivity in fed-batch culture

Background Enhancing the efficiency of process development is a key issue for the production of recombinant therapeutic protein. To maximize both cell mass and productivity with limited time and effort, an improved top-down approach was adopted in the development and optimization of fed-batch culture. Results Two-round optimization was carried out to refine the nutrients following an investigation into the effect of complete feeding medium. After the first round of optimization, cell growth was improved with the integral of viable cell concentration increased by 32.2%. After the second round of optimization, cell productivity was improved with the specific antibody production rate increased 1.5-fold, and cell longevity was also prolonged using improved feeding strategy. In the optimized fed-batch culture in a 2 L bioreactor, the maximum cell concentration was increased by 68% and final antibody titer was increased 3.9-fold compared with the fed-batch culture with simple feeding medium. Conclusion The improved top-down approach helps to reveal the interaction of different nutrients and their important roles in the improvement of cell proliferation and production. Thus, the common defects of a top-down approach can be avoided. This approach is practicable and satisfactory for efficient development of a high-yielding process. © 2013 Society of Chemical Industry

Rapid selection of single cells with high antibody production rates by microwell array

Selection of single cells capable of producing target proteins at high rates is crucial for the development of protein manufacturing processes. Here, we present the rapid selection of single cells producing immunoglobulin antibodies at high specific rates by microwell array and microengraving. Chinese hamster ovary (CHO) cells secreting chimeric antibodies were deposited in a microwell array in a manner such that each microwell contained a single cell. Secreted antibodies in the microwells were transferred onto a glass slide by microengraving, followed by interrogation using fluorescence-based immunoassay. Single cells displaying high signal intensities were selected, retrieved, and clonally expanded to assess their specific antibody production rates. Three successive rounds of the process resulted in the selection of single cells showing significantly increased antibody production rates. The present approach can be applied to the selection of single cells for producing other therapeutic proteins in a high-throughput manner.

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production. The long-term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re-transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational.

Erratum: A nuclear magnetic resonance technique for determining hybridoma cell concentration in hollow fiber bioreactors

We have developed a technique for determining cell concentration in a hollow fiber bioreactor based on 23Na nuclear magnetic resonance (NMR) spectroscopy. Cell concentrations determined with this method agreed closely with concentrations calculated from 31P NMR nucleoside triphosphate (NTP) measurements and oxygen consumption rate measurements. Oxygen transfer limitations, which can complicate cell mass determinations based on oxygen consumption rates, were shown to be negligible for the bioreactor used. Specific antibody production rates in hollow fiber culture, calculated from these cell number estimates, were similar to those found in suspension culture for this cell line.

Responses of GS-NS0 Myeloma cells to osmolality: Cell growth, intracellular mass metabolism, energy metabolism, and antibody production

The influence of osmolality on growth, metabolism, and antibody production of mammalian cells has been widely reported in the past. However, more information about the responses of GS-NS0 Myeloma cells to osmolality, especially regarding the intracellular mass and energy metabolism, has not been available in detail. Fed-batch cultures started at different osmolalities in the range of 280∼370 mOsm/kg were designed to investigate the effects. As the osmolality and cell status changed during the process, cell performance was evaluated in the comparable periods with similar growth rates, nutrition concentrations, and relatively consistent environments. Metabolic flux analysis indicated most of extra consumed glucose at higher osmolalities flowed into lactate formation pathway. The proportion of glucose flux flowed into glycolysis pathway remained approximately 90% and the need of glucose for biomass synthesis was constantly. Also, more than 88% of the glutamine was used in biomass synthesis and the absolute flux remained constant. The specific consumption rate of glutamine declined significantly when cells were cultured in hypo-osmolality (276 mOsm/kg) and a portion of glutamine was synthesized from glutamate. Furthermore, cells were in the state of high energy production at osmolality of 276 mOsm/kg. More glucose flowed into TCA circle with the high efficiency of energy production to meet the demand. Thus, the IVC, the specific antibody production rate, and maximal antibody concentration in fed-batch culture started at 280 mOsm/kg decreased by 35, 36, and 48% compared to those in the culture started at 330 mOsm/kg.

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.

Defined Protein-Free NS0 Myeloma Cell Cultures: Stimulation of Proliferation by Conditioned Medium Factors

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.

A Self-Feeding Roller Bottle for Continuous Cell Culture

The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.

Effects of Synchronization on CD40 Expression and Antibody Production in Hybridoma Cells Stimulated with Anti-mIgG

In previous experiments with the 55-6 hybridoma cell line, we showed that cell stimulation with anti-mouse surface immunoglobulin G antibody (anti-mIgG) increased both CD40 expression and specific monoclonal antibody (mAb) production rate. Cell preincubation with lipopolysaccharide (LPS) prior to anti-mIgG stimulation enhanced these results. Moreover, the expression of both CD40 and surface immunoglobulin G (sIgG) were higher for cells in the G1 phase of the cell cycle. Therefore, to determine the relationship between cell cycle position, CD40 expression, and mAb productivity, in this work cells were synchronized in the G1 phase by thymidine block. In addition, synchronized cells were subjected to different treatments with anti-mIgG. Although synchronized cells showed a slight increase in both CD40 expression and maximum specific growth rate (mu max) compared with unsynchronized cells, specific productivity did not show significant changes. However, the stimulation of synchronized cells with anti-mIgG increased over 65% the expression of CD40 and over 50% the specific productivity in comparison with that obtained on unsynchronized cells after anti-mIgG stimulation. These data improved additionally over 15 and 60%, respectively, by adding 2 mM thymidine to the culture medium. These results suggest that the effect of the positive association between G1 phase, CD40 expression, and specific productivity is subordinated to the effect of anti-mIgG stimulation, which is enhanced by increasing the percentage of cells on the G1 phase of the cell cycle. Contrary to expectations, LPS preincubation of synchronized cells prior to anti-mIgG stimulation did not increase the specific productivity in comparison with non-preincubated cells, and the expression of CD40 was minor compared to that on non-preincubated cells.

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.

Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells

Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d(-1) to 0.2 d(-1). Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d(-1) and increased to 55% when the dilution rate was reduced to 0.2 d(-1). As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d(-1). The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d(-1). By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d(-1). For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.2 d(-1). With an initial increase from 2 to 15 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.4 d(-1), the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d(-1). A good correlation between specific antibody production rate and the percentage of G2/M cells was observed.

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.

A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies

A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines. The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose. The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale. Compared to batch cultures, the fed-batch process yielded a 4. 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration. The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process. For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration. The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1). This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies.