Upstream Development

eBook: Production of Cell-Line Development and Control of Product Consistency During Cultivation — Myths, Risks, and Best Practices

Health authorities are requesting substantial details from sponsors regarding practices used to generate production cell lines for recombinant DNA–(rDNA) derived biopharmaceuticals. Authorities also are asking for information about the clonality of master cell banks (MCBs) and control strategies to minimize genetic heterogeneity. Such requests are prompted by recent reports indicating “nonclonality” for certain production cell lines. To address these and related issues, the CASSS CMC Strategy Forum on “Production Cell Line Development and Control of Product Consistency During Cell Cultivation:…

Cell Expansion with Dissolvable Microcarriers

In recent years we have seen an exponential increase in the number of companies testing and validating new regenerative medicine products. Many of these products are reaching late-phase trials with the potential to receive final approval and marketing authorization from regulatory agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). In the past several years, we have seen successful launches of regenerative medicine products, including Holoclar (Holostem Advanced Therapies), Kymriah (tisagenlecleucel, Novartis), Yescarta…

Enhanced Galactosylation of Monoclonal Antibodies: Using Medium Supplements and Precursors of UDP-Galactose, Part 2

In Part 1 of this report, we described our development of a high-throughput assay for analyzing monoclonal antibody (MAb) glycans and how we used it to evaluate the effects of medium supplements on galactosylation of MAbs produced by two different cell lines (1). This month, we examine galactosylation of a MAb produced by a third cell line. A discussion follows on the benefits of this high-throughput assay before we highlight the similarities and differences in galactosylation among the three MAbs…

Enhanced Galactosylation of Monoclonal Antibodies: Using Medium Supplements and Precursors of UDP-Galactose, Part 1

The biopharmaceutical industry needs better understanding of how monoclonal antibody (MAb) glycosylation is influenced by components in cultivation media — and it needs methods to exert some control over the structure of MAb glycans. That structure can affect MAb function. Thus, a high-throughput (HTP) assay is needed for characterizing MAb glycosylation so that developers can observe the effects of cultivation conditions on MAb glycosylation rapidly, with a goal of producing MAbs that have a desired glycan structure. The method also…

Therapeutic IgG-Like Bispecific Antibodies: Modular Versatility and Manufacturing Challenges, Part 2

Monoclonal antibodies (MAbs) are bivalent and monospecific, with two antigen-binding arms that both recognize the same epitope. Bispecific and multispecific antibodies, collectively referred to herein as bispecific antibodies (bsAbs), can have two or more antigen-binding sites, which are capable of recognizing and binding two or more unique epitopes. Based on their structure, bsAbs can be divided into two broad subgroups: IgG-like bsAbs and non–IgG-like bsAbs. We have chosen to focus on the former in this review. Part one provides a…

eBook: Addressing Quality in Cell-Line Development — Direct Analysis of Bioreactor Harvest for Clone Selection and Process Optimization

Using Direct Analysis of Bioreactor Harvest for Clone Selection and Process Optimization Therapeutic monoclonal antibodies (MAbs) mostly are manufactured using bioengineered mammalian cells cultured in a bioreactor for two to three weeks. High temperatures and an altered redox environment may compromise the quality of MAbs produced (e.g., fragmentation, truncation), as can the presence of proteases, reductases, and other chemicals released from dead cells. Thus, it would be valuable to establish analytical methods that can help cell culture groups monitor immunoglobulin…

An Approach to Generating Better Biosimilars: Considerations in Controlling Glycosylation Variability in Protein Therapeutics

The global market for biotherapeutics has expanded extensively over the past decade and is projected to account for more than a quarter of the pharmaceutical market by 2020, with sales exceeding US$290 billion (1). Continued expansion of the biosimilar marketplace has led to many commercial opportunities and technical challenges. The biological systems used to manufacture such drug products are inherently variable — a feature that has important consequences for the reproducibility, safety, and efficacy of the resulting products. Therefore, a…

Simplify Upstream Process Development and Scale-Up: Single-Use 5:1 Turndown-Ratio Bioreactor Technology

Single-use technologies (SUTs) have been adopted widely in the biopharmaceutical industry for product development as well as clinical- and commercial-scale manufacturing. Over the years, suppliers of such equipment have addressed concerns about waste management, extractables and leachables, and reliability of supply — and as a result, end users have gained confidence in SUTs. Recognizing potential benefits that can be realized for both clinical and commercial operations, biomanufacturers increasingly are implementing SU solutions at larger scales in both upstream production and…

CO2, O2, and Biomass Monitoring in Escherichia coli Shake Flask Culture: Following Glucose–Glycerin Diauxie Online

Carbon dioxide (CO2) is an important parameter in microbial cultures because it can inhibit or stimulate growth under certain conditions. In our experiment, we monitored Escherichia coli diauxie growth phases online and focused on dissolved CO2 (dCO2) and oxygen readings. We assessed diauxic growth in medium containing glycerin and glucose online with the SFR vario system (from PreSens), which optically measures oxygen, pH, and biomass in an Erlenmeyer flask. The shake flask contained an oxygen sensor spot and an optical…

A Stirred, Single-Use, Small-Scale Process Development System: Evaluation for Microbial Cultivation

Mammalian and microbial protein production platforms have been used for over 30 years to produce a number of successful biologic drugs, including monoclonal antibodies (MAbs), recombinant proteins, and therapeutic enzymes (1). Most biologics are produced by mammalian cell lines, with Chinese hamster ovary (CHO) cells being the most widely used. However, microbial cells also are used to express recombinant therapeutic proteins, and almost 30% of currently approved biologics are produced by Escherichia coli bacteria (2). With worldwide biologics sales >56…