Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events.

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns.

N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed. Graphical abstract.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures.

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant ß-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The ß-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased ß-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in ß-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased ß-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

Evaluation of single-use optical and electrochemical pH sensors in upstream bioprocessing.

Culture pH is a critical process parameter during CHO cell bioreactor operations that is key for proper cell growth, protein production, and maintaining the critical quality attributes of a monoclonal antibody drug substance. The traditional means of measuring pH in bioreactors is with an electrochemical probe that can withstand and maintain accuracy through repeated sterilization cycles. An alternative technique for measuring pH is an optical sensor composed of a fluorescent dye that is sensitive to the hydrogen ion concentration. In this work we explore single-use electrochemical and single-use optical pH sensors in stirred-tank and rocking bioreactors, respectively, to understand how their overall performance compares to traditional electrochemical probes in benchtop glass stirred tank bioreactors. We found that the single-use optical pH sensors were generally less accurate than the electrochemical probes, especially in detecting large pH drifts from the setpoint. The single-use electrochemical probes were increasingly accurate as pH was increased from <7.0 to 7.5 but tended to decrease in accuracy as the batch age increased. In conclusion, single-use pH sensors offer a convenient means to measure pH during an upstream bioprocess, but the limitations of these sensors should be built into process control such that deviations in process pH, and consequently potential fluctuations in product quality, can be avoided.

Real time on-line amino acid analysis to explore amino acid trends and link to glycosylation outcomes of a model monoclonal antibody.

Bioreactor parameters can have significant effects on the quantity and quality of biotherapeutics. Monoclonal antibody products have one particularly important critical quality attribute being the distribution of product glycoforms. N-linked glycosylation affects the therapeutic properties of the antibody including effector function, immunogenicity, stability, and clearance rate. Our past work revealed that feeding different amino acids to bioreactors altered the productivity and glycan profiles. To facilitate real-time analysis of bioreactor parameters and the glycosylation of antibody products, we developed an on-line system to pull cell-free samples directly from the bioreactors, chemically process them, and deliver them to a chromatography-mass spectroscopy system for rapid identification and quantification. We were able to successfully monitor amino acid concentration on-line within multiple reactors, evaluate glycans off-line, and extract four principal components to assess the amino acid concentration and glycosylation profile relationship. We found that about a third of the variability in the glycosylation data can be predicted from the amino acid concentration. Additionally, we determined that the third and fourth principal component accounts for 72% of our model's predictive power, with the third component indicated to be positively correlated with latent metabolic processes related to galactosylation. Here we present our work on rapid online spent media amino acid analysis and use the determined trends to collate with glycan time progression, further elucidating the correlation between bioreactor parameters such as amino acid nutrient profiles, and product quality. We believe such approaches may be useful for maximizing efficiency and reducing production costs for biotherapeutics.

Evaluation of association of anti-PEG antibodies with anaphylaxis after mRNA COVID-19 vaccination.

BACKGROUND: The mechanism for anaphylaxis following mRNA COVID-19 vaccination has been widely debated; understanding this serious adverse event is important for future vaccines of similar design. A mechanism proposed is type I hypersensitivity (i.e., IgE-mediated mast cell degranulation) to polyethylene glycol (PEG). Using an assay that, uniquely, had been previously assessed in patients with anaphylaxis to PEG, our objective was to compare anti-PEG IgE in serum from mRNA COVID-19 vaccine anaphylaxis case-patients and persons vaccinated without allergic reactions. Secondarily, we compared anti-PEG IgG and IgM to assess alternative mechanisms. METHODS: Selected anaphylaxis case-patients reported to U.S. Vaccine Adverse Event Reporting System December 14, 2020-March 25, 2021 were invited to provide a serum sample. mRNA COVID-19 vaccine study participants with residual serum and no allergic reaction post-vaccination ("controls") were frequency matched to cases 3:1 on vaccine and dose number, sex and 10-year age category. Anti-PEG IgE was measured using a dual cytometric bead assay (DCBA). Anti-PEG IgG and IgM were measured using two different assays: DCBA and a PEGylated-polystyrene bead assay. Laboratorians were blinded to case/control status. RESULTS: All 20 case-patients were women; 17 had anaphylaxis after dose 1, 3 after dose 2. Thirteen (65 %) were hospitalized and 7 (35 %) were intubated. Time from vaccination to serum collection was longer for case-patients vs controls (post-dose 1: median 105 vs 21 days). Among Moderna recipients, anti-PEG IgE was detected in 1 of 10 (10 %) case-patients vs 8 of 30 (27 %) controls (p = 0.40); among Pfizer-BioNTech recipients, it was detected in 0 of 10 case-patients (0 %) vs 1 of 30 (3 %) controls (p >n 0.99). Anti-PEG IgE quantitative signals followed this same pattern. Neither anti-PEG IgG nor IgM was associated with case status with both assay formats. CONCLUSION: Our results support that anti-PEG IgE is not a predominant mechanism for anaphylaxis post-mRNA COVID-19 vaccination.

Failure Mode Identification of Insulin Drug Products - Impact of Relevant Stress Conditions on the Quality of the Drug.

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.

An etanercept O-glycovariant with enhanced potency.

Most therapeutic proteins are glycosylated with N-glycans and/or O-glycans. N-glycans on therapeutic proteins have been extensively studied for their control strategy and impact on drug product quality. However, knowledge of O-glycosylation in therapeutic protein production and its impact on product quality remains elusive. To address this gap, we generated an O-glycoengineered Chinese Hamster Ovary (CHO) cell line platform to modulate O-glycosylation of therapeutic proteins and investigated the impact of O-glycans on the physicochemical and biological properties of etanercept. Our results demonstrate that this CHO cell line platform produces controlled O-glycosylation profiles containing either truncated O-glycans (sialylTn and/or Tn), or sialylCore 3 alone, or sialylCore 1 with sialylTn or sialylCore 3 O-glycans on endogenous and recombinant proteins. Moreover, the platform demonstrated exclusive modulation of O-glycosylation without affecting N-glycosylation. Importantly, certain O-glycans on etanercept enhanced tumor necrosis factor-α binding affinity and consequent potency. This is the first report that describes the systematic establishment of an O-glycoengineered CHO cell line platform with direct evidence that supports the applicability of the platform in the production of engineered proteins with desired O-glycans. This platform is valuable for identifying O-glycosylation as a critical quality attribute of biotherapeutics using the quality by design principle.

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

Physico-mechanical and stability evaluation of carbamazepine cocrystal with nicotinamide.

The focus of this investigation was to prepare the cocrystal of carbamazepine (CBZ) using nicotinamide as a coformer and to compare its preformulation properties and stability profile with CBZ. The cocrystal was prepared by solution cooling crystallization, solvent evaporation, and melting and cryomilling methods. They were characterized for solubility, intrinsic dissolution rate, chemical identification by Fourier transform infrared spectroscopy, crystallinity by differential scanning calorimetry, powder X-ray diffraction, and morphology by scanning electron microscopy. Additionally, mechanical properties were evaluated by tensile strength and Heckel analysis of compacts. The cocrystal and CBZ were stored at 40°C/94% RH, 40°C/75% RH, 25°C/60% RH, and 60°C to determine their stability behavior. The cocrystals were fluffy, with a needle-shaped crystal, and were less dense than CBZ. The solubility profiles of the cocrystals were similar to CBZ, but its intrinsic dissolution rate was lower due to the high tensile strength of its compacts. Unlike CBZ, the cocrystals were resistant to hydrate transformation, as revealed by the stability studies. Plastic deformation started at a higher compression pressure in the cocrystals than CBZ, as indicated by the high yield pressure. In conclusion, the preformulation profile of the cocrystals was similar to CBZ, except that it had an advantageous resistance to hydrate transformation.

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator.

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

High Performance Size Exclusion Chromatography and High-Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products.

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10-12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1-1000 nm) in the submicron size range.

Multivariate data analysis of growth medium trends affecting antibody glycosylation.

Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data-generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in-house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N-glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation. Due to the amount of in-process data generated by PAT instrumentation, multivariate data analysis was necessary to ascertain which variables best modeled our glycan profile findings. Our principal component analysis revealed components that represent the development of glycoforms into terminally galacotosylated forms (G1F and G2F), and another that encompasses maturation out of high mannose glycoforms. The partial least squares model additionally incorporated metabolic values to link these processes to glycan outcomes, especially involving the consumption of glutamine. Overall, these approaches indicated a tradeoff between cellular productivity and product quality in terms of the glycosylation. This work illustrates the use of multivariate analytical approaches that can be applied to complex bioprocessing problems for identifying potential solutions.

The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

An ICP-MS platform for metal content assessment of cell culture media and evaluation of spikes in metal concentration on the quality of an IgG3:κ monoclonal antibody during production.

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.

The Current Scientific and Regulatory Landscape in Advancing Integrated Continuous Biopharmaceutical Manufacturing.

There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. On the upstream side of biotechnology manufacturing, continuous perfusion cell cultures are fairly well established. However, truly integrated continuous biomanufacturing requires the uninterrupted connection of continuous unit operations (upstream and downstream) with no isolated intermediate or hold steps occurring between them. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing.

Real-time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality.

Real-time monitoring of cell cultures in bioreactors can enable expedited responses necessary to correct potential batch failure perturbations which may normally go undiscovered until the completion of the batch and result in failure. Currently, analytical technologies are dedicated to real-time monitoring of bioreactor parameters such as pH, dissolved oxygen, and temperature, nutrients such as glucose and glutamine, or metabolites such as lactate. Despite the importance of amino acids as the building blocks of therapeutic protein products, other than glutamine their concentrations are not commonly measured. Here, we present a study into amino acid monitoring, supplementation strategies, and how these techniques may impact the cell growth profiles and product quality. We used preliminary bioreactor runs to establish baselines by determining initial amino acid consumption patterns, the results of which were used to select a pool of amino acids which gets depleted in the bioreactor. These amino acids were combined into blends which were supplemented into bioreactors during a subsequent run, the concentrations of which were monitored using a mass spectrometry based at-line method we developed to quickly assess amino acid concentrations from crude bioreactor media. We found that these blends could prolong culture life, reversing a viable cell density decrease that was leading to batch death. Additionally, we assessed how these strategies might impact protein product quality, such as the glycan profile. The amino acid consumption data were aligned with the final glycan profiles in principal component analysis to identify which amino acids are most closely associated with glycan outcomes.

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.