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Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed‐batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed‐batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:793–805, 2018 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016 [ABSTRACT FROM AUTHOR]

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules. [ABSTRACT FROM AUTHOR]

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile. [ABSTRACT FROM AUTHOR]

BACKGROUND In mammalian cell culture processes, viable cell density ( VCD) is one of the most crucial parameters for monitoring of the current status of cell growth and protein production. Among the various real-time monitoring tools available for VCD in Chinese hamster ovary ( CHO) cell culture, dielectric spectroscopy is one of the more reliable due to its simplicity, robustness and accuracy. These attributes enable efficient monitoring and control of the corresponding processes. However, one of the major drawbacks of dielectric spectroscopy is frequent deterioration of accuracy during the late-growth phase of CHO cell culture. This can be caused by abrupt changes in the cell physiology, as well as environmental conditions. RESULTS In this study, various kinds of chemometric tools were employed to understand the spectral dynamics of dielectric spectroscopy during extended CHO cell culture, including different cell growth phases. Then, the performance of different multivariate regression models were compared with conventional Cole-Cole equation-based approaches. The models were compared in their ability to estimate VCD in CHO cell culture from dielectric spectra from several batches of bioreactor operations. Multivariate analysis of dielectric spectral datasets clearly revealed the time-varying evolution of spectral dynamics during the transition between cell growth phases. The best prediction performance from the dielectric spectroscopy was realized using a regression model based on locally-weighted partial least squares ( LWPLS). This estimation model best compensated for spectral dynamics observed during the different cell growth phases. CONCLUSION Dielectric spectroscopy combined with LWPLS allows achievement of optimal and stable estimation of VCD during CHO cell culture, regardless of inherent differences in cell growth phases. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are biological macromolecules with complex post-translational modifications that can be observed when assessing product variants. The N- and C-terminal heterogeneities of commercially produced antibodies have been observed and extensively studied over the past 30 years. This review summarizes the current literature on detectable antibody termini variants from cultured cells. The presence of these heterogeneities can be detected by many different analytical methods, mostly based on sequence, charge and size differences. Examples are presented that highlight terminal heterogeneities, methods of detection, and their impact on the quality of mAbs. Regulatory considerations are also discussed regarding the potential impact on product quality, safety, and efficacy. [ABSTRACT FROM AUTHOR]

Currently, there is a gap in the knowledge of the culture responses to controlled bioreactor environment during the course of batch cell culture from early exponential phase to stationary-phase. If available, such information could be used to designate gene transcripts for predicting cell status and as a quality predictor for a controlled bioreactor. In this study, we used oligonucleotide microarrays to obtain baseline gene expression profiles during the time-course of a hybridoma batch cell culture in a 5 L bench-scale bioreactor. Gene expression changes that were up or down modulated from early-to-late in batch culture, as well as invariant gene profiles with significant expression were identified using microarray. Typical cellular functions that seemed to be correlated with transcriptomics were oxidative stress response, DNA damage response, apoptosis, and cellular metabolism. As confirmatory evidence, microarray findings were verified with a more rigorous semiquantitative gene-specific Reverse transcriptase-polymerase chain reaction (RT-PCR). The results of this study suggest that under predefined bioreactor culture conditions, significant gene changes from lag to log to stationary phase could be identified, which could then be used to track the culture state. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 [ABSTRACT FROM AUTHOR]

Scale-down of bioreactors is currently done based on matching one or more measurable parameters such as kL a and P/ V, which could result in insufficient process comparability. Currently, there is a lack of genomic translational studies in cell culture scale-down, which could help delineate measurable cellular attributes for improved scale-down. In this study, we scaled-down from a typical bench-scale 5-L bioreactor to a novel high-throughput 35-mL minibioreactor based on matching oxygen transfer rate, which resulted in cell growth and product-related discrepancies using Sp2/0 cells. Performing DNA microarrays on time-course samples from both systems, we identified ∼200 differentially expressed transcripts, presumably because of bioreactor aeration and mixing differences with scale-down. Evaluating these transcripts for bioreactor-relevant cellular functions such as oxidative stress response and DNA damage response, we chose 18 sentinel genes based on their degree of difference and functionality, which we further verified by quantitative real-time polymerase chain reaction ( qRT-PCR). Tracking the differential expression of Sod1, Apex1, and Odc1 genes, we were able to correlate sparging-related damage and poor mixing, as possible causes for physiological changes such as prolonged culture in minibioreactors. Additionally, to verify our sentinel gene findings, we performed follow-up improved scale-down studies based on gene analysis and measured transcriptomic changes. As a result, qRT-PCR-based genomic profiles and cell growth profiles showed better convergence between the improved minibioreactor conditions and the model 5-L bioreactor. Our results broadly show that based on the knowledge from transcriptomic changes of sentinel gene profiles, it is possible to improve bioreactor scale-down for more comparable processes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 [ABSTRACT FROM AUTHOR]

The bioprocess development cycle is a complex task that requires a complete understanding of the engineering of the process (e.g., mass transfer, mixing, CO removal, process monitoring, and control) and its affect on cell biology and product quality. Despite their widespread use in bioprocess development, spinner flasks generally lack engineering characterization of critical physical parameters such as k a, P/ V, or mixing time. In this study, mass transfer characterization of a 250-mL spinner flask using optical patch-based sensors is presented. The results quantitatively show the effect of the impeller type, liquid filling volume, and agitation speed on the volumetric mass transfer coefficient ( k a) in a 250-mL spinner flask, and how they can be manipulated to match mass transfer capability at large culture devices. Thus, process understanding in spinner flasks can be improved, and these devices can be seamlessly integrated in a rational scale-up strategy from cell thawing to bench-scale bioreactors (and beyond) in biomanufacturing. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011. [ABSTRACT FROM AUTHOR]

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench-scale is limited by the scale-up and operational costs involved. This study describes the application of miniaturized stirred high-throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Retrovirus-like particles (RVLPs) that are expressed during the production of monoclonal antibodies in Chinese hamster ovary (CHO) cell cultures must be removed during product recovery. Anion exchange chromatography (AEX) performed in product flow-through mode, a common component in the purification of monoclonal antibodies, has been shown to provide robust removal of a related retrovirus model, but it's ability to remove the actual RVLP impurities has not been directly investigated. We have determined the ability of a typical Q sepharose process to remove actual CHO RVLP impurities. Using small scale experiments with three model antibodies, we observe that this AEX process is capable of effectively removing both in-process and spiked RVLPs from different feedstocks containing different mAb products. In addition, we show that this AEX process also achieves a similarly high degree of RVLP removal during large scale manufacturing operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

The article discusses a study which compared optical pH and dissolved oxygen sensors with the traditional electrochemical probes used in mammalian cell culture. The Pearson Correlations revealed the optical sensors have 98.7 percent while dissolved oxygen have 99.7 percent with a difference in pH media of 0.04 pH units. The study showed that optical probes exhibited accuracy in detecting the potential parameter drift which would impact the production kinetics, growth and production quality.

Monoclonal antibodies (mAbs) are commonly glycosylated and show varying levels of galactose attachment. A set of experiments in our work showed that the galactosylation level of mAbs was altered by the culture conditions of hybridoma cells. The uridine diphosphate galactose (UDP-Gal) is one of the substrates of galactosylation. Based on that, we proposed a two-step model to predict N-linked glycoform profiles by solely using extracellular metabolites from cell culture. At the first step, the flux level of UDP-Gal in each culture was estimated based on a computational flux balance analysis (FBA); its level was found to be linear with the galactosylation degree on mAbs. At the second step, the glycoform profiles especially for G0F (agalactosylated), G1F (monogalactosylated) and G2F (digalactosylated) were predicted by a kinetic model. The model outputs well matched with the experimental data. Our study demonstrated that the integrated mathematical approach combining FBA and kinetic model is a promising strategy to predict glycoform profiles for mAbs during cell culture processes. [ABSTRACT FROM AUTHOR]

Highlights • An ICP-MS methodology to determine bulk, minor and trace metal content (0.05 ppb–500 ppb) of mammalian cell culture media. • An advanced autodilution platform for autocalibration and quality control to enhance throughput. • Application of this method to demonstrate the affected homogeneity of a model therapeutic due to spikes in the concentration of copper and iron in parallel bioreactors. Abstract Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced. [ABSTRACT FROM AUTHOR]

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett- Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, β-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1919-1928, 2015 [ABSTRACT FROM AUTHOR]

Abstract: Cell culture optimization is a labor-intensive process requiring a large number of experiments to be conducted under varying conditions. Here we describe a high-throughput bioreactor system that allows 12 mini stirred-tank bioreactors to be operated simultaneously. All bioreactors are monitored by low-cost minimally invasive optical sensors for pH and dissolved oxygen. The sensors consist of single-use patches affixed inside the bioreactors and monitored optically from the outside. Experimental results show that different sensing patches with the same composition respond consistently. The discrepancy between different pH sensors is less than 0.1 pH units over most of their responsive range. The discrepancy between different dissolved oxygen sensors is less than 10% over the whole range from 0% to 100% dissolved oxygen. The consistency of the sensing system ensures that only an initial one-time calibration is required for the sensing patches. After that, a calibration code is generated and sensing patches of the same composition can be used directly. This greatly reduces the time and cost required for monitored multi-bioreactor operations. We used SP2/0 myeloma/mouse hybridoma cell cultures to demonstrate reactor performance consistency. Transcriptional profiling, HPLC analysis, viable cell count, and viability inspection show that the presence of sensing patches and the use of optical monitoring have no apparent effect on the metabolism of the cells. [Copyright &y& Elsevier]