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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Investigation of metabolic variability observed in extended fed batch cell culture.

A 13-day fed-batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell-specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions.

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance.

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture.

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable.

Characterization of trisulfide modification in antibodies.

Trisulfides are a posttranslational modification formed by the insertion of a sulfur atom into a disulfide bond. Although reports for trisulfides in proteins are limited, we find that they are a common modification in natural and recombinant antibodies of all immunoglobulin G (IgG) subtypes. Trisulfides were detected only in interchain linkages and were predominantly in the light-heavy linkages.