Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.

Metabolic rewiring revealed by cell-specific rate analyses from nontargeted exometabolomics during simultaneous consumption of glucose and lactic acid in a CHO fed-batch process.

Previously, we reported, based on an untargeted metabolomics, carnitine derivatives are part of a mechanism to overcome impaired mitochondrial functioning triggered by an acyl-group overflow in CHO cells. In this study, we analyzed the cell-specific rates of 24 selected metabolites using two metrics: correlation coefficients and root-mean-square deviations (RMSDs) between glucose-fed versus glucose/lactic acid-fed cultures. The time-course profiles of acetylcarnitine, adipoylcarnitine, glutarylcarnitine, glutamate, and succinate exhibited significant negative correlations between the two culture conditions. Based on RMSDs, seven carnitine derivatives, 3-hydroxy-methyl-glutarate, mevalonate, pyridoxamine-5-phosphate, succinate, and glycine were substantially different. The analyses from the two metrics reveal a distinctive rearrangement of rates from the following metabolic pathways: (i) high secretion rates of carnitines as part of the acyl-group removal, (ii) low secretion rates of succinate, related to the tricarboxylic acid cycle and the electron-transport chain, (iii) low secretion rates of pyridoxamine-5-phosphate - a co-factor for amino acid catabolism, transaminations, and transsulfuration, and (iv) increases in the consumption rates of glutamate and glycine, both used to produce glutathione. The rewiring in rates observed upon feeding lactic acid is best explained by the activation of pathways supporting homeostasis of acyl-groups and antioxidant synthesis, which are required for continuous proper functioning of oxidative phosphorylation.

A Fully Integrated Online Platform For Real Time Monitoring Of Multiple Product Quality Attributes In Biopharmaceutical Processes For Monoclonal Antibody Therapeutics.

In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.

Characterization of cell cycle and apoptosis in Chinese hamster ovary cell culture using flow cytometry for bioprocess monitoring.

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.

Exometabolome profiling reveals activation of the carnitine buffering pathway in fed-batch cultures of CHO cells co-fed with glucose and lactic acid.

Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy-based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi-quantitative and time-dependent analysis of the exometabolome. This study focused on the CHO cell's metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon-buffering pathway. CHO cells exhibited a balance between intermediates from (i) amino acid catabolism, (ii) fatty acid ß-oxidation, and (iii) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3-hydroxy-methyl-glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl-CoA groups as well as an ability to maintain the acyl-CoA: free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine-buffering system, HMG, and mevalonate pathways.

Tuning monoclonal antibody galactosylation using Raman spectroscopy-controlled lactic acid feeding.

A key aspect of large-scale production of biotherapeutics is a well-designed and consistently-executed upstream cell culture process. Process analytical technology tools provide enhanced monitoring and control capabilities to support consistent process execution, and also have potential to aid in maintenance of product quality at desired levels. One such tool, Raman spectroscopy, has matured as a useful technique to achieve real-time monitoring and control of key cell culture process attributes. We developed a Raman spectroscopy-based nutrient control strategy to enable dual control of lactate and glucose levels for a fed-batch CHO cell culture process for monoclonal antibody (mAb) production. To achieve this, partial least squares-based chemometric models for real-time prediction of glucose and lactate concentrations were developed and deployed in feedback control loops. In particular, feeding of lactic acid post-metabolic shift was investigated based on previous work that has shown the impact of lactate levels on ammonium as well as mAb product quality. Three feeding strategies were assessed for impact on cell metabolism, productivity, and product quality: bolus-fed glucose, glucose control at 4 g/L, or simultaneous glucose control at 4 g/L and lactate control at 2 g/L. The third feeding strategy resulted in a significant reduction in ammonium levels (68%) while increasing mAb galactosylation levels by approximately 50%. This work demonstrated that when deployed in a cell culture process, Raman spectroscopy is an effective technique for simultaneous control of multiple nutrient feeds, and that lactic acid feeding can have a positive impact on both cell metabolism and mAb product quality.

Simultaneous Monitoring and Comparison of Multiple Product Quality Attributes for Cell Culture Processes at Different Scales Using a LC/MS/MS Based Multi-Attribute Method.

In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.

Omega-3-Fortified Lipid-Based Nutrient Supplement: Development, Characterization, and Consumer Acceptability.

BACKGROUND: Incorporation of omega-3 polyunsaturated fatty acid (PUFA) sources to lipid-based nutrition supplement (LNS) formulations for undernourished populations presents a challenge due to reduced shelf life and poor consumer acceptability. OBJECTIVE: To systematically develop an omega-3-fortified LNS formulation, using flaxseed oil (FO), by optimizing its processing stability and evaluating its sensory acceptance. METHODS: LNS formulations were formulated to yield approximately 452 kcal, 13.2 g of protein, 30 g of fat, and 53 g of carbohydrates per 100 g. Response surface methodology (RSM) with 4-factor-3-level: omega-3 fatty acid source (FO; 0%-10%), antioxidant (ascorbyl palmitate: 0.0%-0.03%), emulsifier (soy lecithin: 0.5%-1.5%), and storage time (0-6 months at 40°C) was used to optimize LNS's functionality and storage stability. Factor effects were evaluated for peroxide value, oil separation, water activity, moisture content, hardness, and vitamin C content of the samples. Consumer acceptability was assessed using a 9-point hedonic scale. RESULTS: After 6 months of accelerated storage, the addition of FO to LNS formula resulted in increased peroxide values (7.75 mEq/kg fat) and lipid separation and a reduction in vitamin C and water activity. Optimal levels to maximize storage time and FO and minimize oxidation were 0.02% antioxidant, 1.5% emulsifier, and 4.9% FO. Indian women and students accepted LNS formulations with or without FO similar to other commercial supplements. CONCLUSION: An LNS containing FO with improved omega-6/omega-3 PUFA ratio was successfully formulated with staple Indian ingredients and optimized for storage stability using RSM.

An Integrated Solution-Based Rapid Sample Preparation Procedure for the Analysis of N-Glycans From Therapeutic Monoclonal Antibodies.

Consistent glycosylation in therapeutic monoclonal antibodies is a major concern in the biopharmaceutical industry as it impacts the drug's safety and efficacy and manufacturing processes. Large numbers of samples are created for the analysis of glycans during various stages of recombinant proteins drug development. Profiling and quantifying protein N-glycosylation is important but extremely challenging due to its microheterogeneity and more importantly the limitations of existing time-consuming sample preparation methods. Thus, a quantitative method with fast sample preparation is crucial for understanding, controlling, and modifying the glycoform variance in therapeutic monoclonal antibody development. Presented here is a rapid and highly quantitative method for the analysis of N-glycans from monoclonal antibodies. The method comprises a simple and fast solution-based sample preparation method that uses nontoxic reducing reagents for direct labeling of N-glycans. The complete work flow for the preparation of fluorescently labeled N-glycans takes a total of 3 h with less than 30 min needed for the release of N-glycans from monoclonal antibody samples.