Using Metabolomics to Identify Cell Line-Independent Indicators of Growth Inhibition for Chinese Hamster Ovary Cell-based Bioprocesses.

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals. Efforts to improve productivity through medium design and feeding strategy optimization have focused on preventing the depletion of essential nutrients and managing the accumulation of lactate and ammonia. In addition to ammonia and lactate, many other metabolites accumulate in CHO cell cultures, although their effects remain largely unknown. Elucidating these effects has the potential to further improve the productivity of CHO cell-based bioprocesses. This study used untargeted metabolomics to identify metabolites that accumulate in fed-batch cultures of monoclonal antibody (mAb) producing CHO cells. The metabolomics experiments profiled six cell lines that are derived from two different hosts, produce different mAbs, and exhibit different growth profiles. Comparing the cell lines' metabolite profiles at different growth stages, we found a strong negative correlation between peak viable cell density (VCD) and a tryptophan metabolite, putatively identified as 5-hydroxyindoleacetaldehyde (5-HIAAld). Amino acid supplementation experiments showed strong growth inhibition of all cell lines by excess tryptophan, which correlated with the accumulation of 5-HIAAld in the culture medium. Prospectively, the approach presented in this study could be used to identify cell line- and host-independent metabolite markers for clone selection and bioprocess development.

The importance and future of biochemical engineering.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

Multi-Omics Reveals Impact of Cysteine Feed Concentration and Resulting Redox Imbalance on Cellular Energy Metabolism and Specific Productivity in CHO Cell Bioprocessing.

Chinese hamster ovary (CHO) cells are currently the primary host cell lines used in biotherapeutic manufacturing of monoclonal antibodies (mAbs) and other biopharmaceuticals. Cellular energy metabolism and endoplasmic reticulum (ER) stress are known to greatly impact cell growth, viability, and specific productivity of a biotherapeutic; but the molecular mechanisms are not fully understood. The authors previously employed multi-omics profiling to investigate the impact of a reduction in cysteine (Cys) feed concentration in a fed-batch process and found that disruption of the redox balance led to a substantial decline in cell viability and titer. Here, the multi-omics findings are expanded, and the impact redox imbalance has on ER stress, mitochondrial homeostasis, and lipid metabolism is explored. The reduced Cys feed activates the amino acid response (AAR), increases mitochondrial stress, and initiates gluconeogenesis. Multi-omics analysis reveals that together, ER stress and AAR signaling shift the cellular energy metabolism to rely primarily on anaplerotic reactions, consuming amino acids and producing lactate, to maintain energy generation. Furthermore, the pathways are demonstrated in which this shift in metabolism leads to a substantial decline in specific productivity and altered mAb glycosylation. Through this work, meaningful bioprocess markers and targets for genetic engineering are identified.

CHO genome mining for synthetic promoter design.

Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. CHO genes varying in transcriptional activity were selected based on a comparative analysis of RNA-Seq transcript levels in normal and biphasic cultures in combination with estimates of mRNA half-life from published genome scale datasets. Discrete transcription factor regulatory elements (TFREs) upstream of these genes were informatically identified and functionally screened in vitro to identify a subset of TFREs with the potential to support high activity recombinant gene transcription during biphasic cell culture processes. Two libraries of heterotypic synthetic promoters with varying TFRE combinations were then designed in silico that exhibited a maximal 2.5-fold increase in transcriptional strength over the CMV-IE promoter after transient transfection into host CHO-K1 cells. A subset of synthetic promoters was then used to create stable transfectant pools using CHO-K1 cells under glutamine synthetase selection. Whilst not achieving the maximal 2.5-fold increase in productivity over stable pools harboring the CMV promoter, all stably transfected cells utilizing synthetic promoters exhibited increased reporter production - up to 1.6-fold that of cells employing CMV, both in the presence or absence of intron A immediately downstream of the promoter. The increased productivity of stably transfected cells harboring synthetic promoters was maintained during fed-batch culture, with or without a transition to mild hypothermia at the onset of stationary phase. Our data exemplify that it is important to consider both host cell and intended bioprocess contexts as design criteria in the de novo construction of synthetic genetic parts for mammalian cell engineering.

Multi-Omics Study on the Impact of Cysteine Feed Level on Cell Viability and mAb Production in a CHO Bioprocess.

There is continual demand to maximize CHO cell culture productivity of a biotherapeutic while maintaining product quality. In this study, a comprehensive multi-omics analysis is performed to investigate the cellular response to the level of dosing of the amino acid cysteine (Cys) in the production of a monoclonal antibody (mAb). When Cys feed levels are insufficient, there is a significant decrease in protein titer. Multi-omics (metabolomics and proteomics, with support from RNAseq) is performed over the time course of the CHO bioprocess producing an IgG1 mAb in 5 L bioreactors. Pathway analysis reveals that insufficient levels of Cys in the feed lead to Cys depletion in the cell. This depletion negatively impacts antioxidant molecules, such as glutathione (GSH) and taurine, leading to oxidative stress with multiple deleterious cellular effects. In this paper, the resultant ER stress and subsequent apoptosis that affects cell viability and viable cell density has been considered. Key metabolic enzymes and metabolites are identified that can be potentially monitored as the process progresses and/or increased in the cell either by nutrient feeding or genetic engineering. This work reinforces the centrality of redox balance to cellular health and success of the bioprocess as well as the power of multi-omics to provide an in-depth understanding of the CHO cell biology during biopharmaceutical production.

Glucose monitoring and adaptive feeding of mammalian cell culture in the presence of strong autofluorescence by near infrared Raman spectroscopy.

Raman spectroscopy offers an attractive platform for real-time monitoring and control of metabolites and feeds in cell culture processes, including mammalian cell culture for biopharmaceutical production. However, specific cell culture processes may generate substantial concentrations of chemical species and byproducts with high levels of autofluorescence when excited with the standard 785 nm wavelength. Shifting excitation further toward the near-infrared allows reduction or elimination of process autofluorescence. We demonstrate such a reduction in a highly autofluorescent mammalian cell culture process. Using the Kaiser RXN2-1000 platform, which utilizes excitation at 993 nm, we developed multivariate glucose models in a cell culture process which was previously impossible using 785 nm excitation. Additionally, the glucose level in the production bioreactor was controlled entirely by Raman adaptive feeding, allowing for maintenance of glucose levels at an arbitrary set point for the duration of the culture. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1574-1580, 2018.

Innovation in Cell Banking, Expansion, and Production Culture.

Cell culture-based production processes enable the development and commercial supply of recombinant protein products. Such processes consist of the following elements: thaw and initiation of culture, seed expansion, and production culture. A robust cell source storage system in the form of a cell bank is developed and cells are thawed to initiate the cell culture process. Seed culture expansion generates sufficient cell mass to initiate the production culture. The production culture provides an environment where the cells can synthesize the product and is optimized to deliver the highest possible product concentration with acceptable product quality. This chapter describes the significant innovations made in these process elements and the resulting improvements in the overall efficiency, robustness, and safety of the processes and products.

Lipidomics of CHO Cell Bioprocessing: Relation to Cell Growth and Specific Productivity of a Monoclonal Antibody.

As the demand for biological therapeutic proteins rises, there is an increasing need for robust and highly efficient bioprocesses, specifically, maximizing protein production by controlling the cellular nutritional and metabolic needs. A comprehensive lipidomics analysis has been performed, for the first time, over the time course of CHO cells producing an IgG1 monoclonal antibody (mAb) with fed batch 5 L bioreactors. The dynamic nature and importance of the CHO lipidome, especially on cellular growth and specific productivity, is demonstrated. A robust LC-MS method using positive and negative mode ESI was developed for lipid identification and quantitation of 377 unique lipids. The analysis revealed large changes in lipid features between the different days in bioprocessing including accumulation of triacylglycerol (TG) and lysophospholipid species with depletion of diacylglycerol (DG) species. Exploring pathway analysis where the lipid data was combined with polar metabolites and transcriptomics (RNA sequencing) revealed differences in lipid metabolism between the various stages of cellular growth and highlighted the role of key features of lipid metabolism on cell growth and specific productivity. The study demonstrates the importance of lipidomics in the expanding role of 'Omics methodologies in gaining insight into cellular behavior during protein production in a fed batch bioprocess.

Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic.

The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a "fast-to-tox" strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1468-1475, 2017.

Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives.

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.

Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity.

Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB's high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB's cost, scale-up, and operability challenges.

Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture.

Mitigating risks to biotherapeutic protein production processes and products has driven the development of targeted process analytical technology (PAT); however implementing PAT during development without significantly increasing program timelines can be difficult. The development of a monoclonal antibody expressed in a Chinese hamster ovary (CHO) cell line via fed-batch processing presented an opportunity to demonstrate capabilities of altering percent glycated protein product. Glycation is caused by pseudo-first order, non-enzymatic reaction of a reducing sugar with an amino group. Glucose is the highest concentration reducing sugar in the chemically defined media (CDM), thus a strategy controlling glucose in the production bioreactor was developed utilizing Raman spectroscopy for feedback control. Raman regions for glucose were determined by spiking studies in water and CDM. Calibration spectra were collected during 8 bench scale batches designed to capture a wide glucose concentration space. Finally, a PLS model capable of translating Raman spectra to glucose concentration was built using the calibration spectra and spiking study regions. Bolus feeding in mammalian cell culture results in wide glucose concentration ranges. Here we describe the development of process automation enabling glucose setpoint control. Glucose-free nutrient feed was fed daily, however glucose stock solution was fed as needed according to online Raman measurements. Two feedback control conditions were executed where glucose was controlled at constant low concentration or decreased stepwise throughout. Glycation was reduced from ∼9% to 4% using a low target concentration but was not reduced in the stepwise condition as compared to the historical bolus glucose feeding regimen.

Tryptophan oxidation catabolite, N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance.

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.

Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.

High-throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high-throughput mini-bioreactor system viz. the Advanced Microscale Bioreactor (ambr15(TM) ), to perform process characterization in less than a month and develop an input control strategy. As a pre-requisite to process characterization, a scale-down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO2 profiles as well as several other process and product quality parameters. The scale-down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15(TM) system is capable of replacing the bench scale bioreactor system for routine process development and process characterization.

Advanced process monitoring and feedback control to enhance cell culture process production and robustness.

It is a common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds, based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply process analytical technology (PAT), a fully automated cell culture feedback control system has been explored in three different applications. The first study illustrates that frequent monitoring and automatically controlling the complex feed based on a surrogate (glutamate) level improved protein production. More importantly, the resulting feed strategy was translated into a manufacturing-friendly manual feed strategy without impact on product quality. The second study demonstrates the improved process robustness of an automated feed strategy based on online bio-capacitance measurements for cell growth. In the third study, glucose and lactate concentrations were measured online and were used to automatically control the glucose feed, which in turn changed lactate metabolism. These studies suggest that the auto-feedback control system has the potential to significantly increase productivity and improve robustness in manufacturing, with the goal of ensuring process performance and product quality consistency.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

Integration of cell line and process development to overcome the challenge of a difficult to express protein.

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi-prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench-top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10-fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening.

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality.

Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.