Analytical

Evolving Bioassay Strategies for Therapeutic Antibodies: Essential Information for Proving Biosimilarity

The modern age of biologics began 35 years ago with the approval of Lilly’s Humulin product — a biosynthetic form of human insulin derived from recombinant DNA and microbial cell culture (1). Today, about a quarter (27%) of the drugs approved yearly by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) are biopharmaceuticals: primarily monoclonal antibodies (MAbs), but also vaccines, blood products, and (recently), advanced therapies based on genes and cells. A decade ago, the average…

Biological Characterization Using Protein Crystal Measurements

Monoclonal antibodies (MAbs) have become important therapies and are projected to generate US$125 billion in sales by 2020 (1). Given that potential revenue, biopharmaceutical companies are highly motivated to find novel ways to deliver their drug products and extend patent lifetimes. Many such therapies are administered intravenously at low concentration and large volumes (2). Althea’s Crystalomics technology facilitates delivery of traditional biotherapeutics with high concentration, low viscosity, and low volume by arranging a drug’s individual molecules in an ordered crystal…

Comparing Culture Methods in Monoclonal Antibody Production: Batch, Fed-Batch, and Perfusion

Recombinant protein manufacturing with Chinese hamster ovary (CHO) cells represents over 70% of the entire biopharmaceutical industry (1). In fact, human monoclonal antibodies (hMAbs) produced by CHO cells have played a major role in both the diagnostic and therapeutic markets for decades. One of the first human–mouse chimeric MAbs to obtain FDA approval was Roche’s rituximab treatment for non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, and rheumatoid arthritis. Since that approval in 1997, scores of chimeric, humanized, and human MAbs have gained…

Conditional/Inducible Gene-Expression Mouse Models Using Advanced Gene Editing

Transgenic mouse models have been an essential part of biomedical research for many decades. They have provided valuable insights in developmental biology, gene regulation, and our understanding of the genetic basis of human disease. And they play a critical role in drug discovery and development. Traditional methods to generate these mouse models entailed a milieu of disadvantages: e.g., low efficiency, high incidence of undesirable recombination outcomes, randomly and multiply inserted genes of interest, ectopic expression, gene silencing, and insertional mutations…

Residual Host-Cell DNA in Biopharmaceutical Products: 96-Well Plate-Based Extraction and Real-Time PCR Assay for Quantitative Measurement

Regulatory guidelines that cover the development of biopharmaceutical products require testing of host-cell deoxyribonucleic acid (DNA) impurities. Real-time polymerase chain reaction (PCR) has become a popular technology for DNA quantitation and monitoring of process impurities associated with biomanufacturing. One critical challenge associated with host-cell DNA impurity testing is that recombinant proteins (e.g., monoclonal antibodies, MAbs) and their corresponding buffer components often interfere with DNA quantitation in real-time PCR reactions (1, 2). Some sample types do not require a full extraction…

Response to the Publication of USP ‹1207›

The BioPhorum Operation Group’s (BPOG’s) Container Closure Integrity Testing (CCIT) workstream would like to congratulate the United States Pharmacopeia’s committee for its latest revision to USP chapter <1207> Package Integrity Evaluation: Sterile Products. Generally, we believe it provides a comprehensive overview of the available methods for container–closure testing and outlines many important elements for consideration in establishing a successful CCIT strategy. We first responded to the USP <1207> draft when it was released for comment in 2014. And from our…

HCP Antigens and Antibodies from Different CHO Cell Lines

Cell lines derived from Chinese hamster ovary (CHO) cells are widely used in therapeutic protein production because they can perform human-compatible posttranslational modifications, they are easy to use for manufacturing, and they do not propagate most human pathogenic viruses (1, 2). Expressed therapeutic proteins are secreted into CHO culture supernatant along with impurities originating from the host cells themselves. Such host cell proteins (HCPs) are important contaminants for monitoring because they directly affect drug quality, safety, and efficacy. HCPs are…

Special Report on Process- and Product-Related Impurities (A CMC Strategy Forum Special Focus Series): Extractables, Leachables, Particles, and Aggregates

The CMC Strategy Forums focus on relevant chemistry, manufacturing, and controls (CMC) issues throughout the life cycle of a therapeutic and thereby foster collaborative technical and regulatory interaction. Forum chairs share information with regulatory agencies to help them merge good scientific and regulatory practices. Outcomes of forum meetings are published in BioProcess International and on the CASSS website (www.casss.org). This process is meant to help ensure that biopharmaceutical products manufactured with advancing technologies in a regulated environment will continue to…

Mass Spectrometric Conjugate Characterization: Process Qualification of Recombinant Protein–Hapten Conjugation

Conjugated protein biotherapeutics such as PEGylated proteins (with polyethylene glycol), antibody–drug conjugates (ADCs), and protein–haptens often present unique analytical challenges related to characterizing the conjugation aspect of their manufacturing processes. Analytical characterization of this class of proteins requires knowledge of the sites of conjugation, the degree of conjugation, and the drug-to-protein ratio. Here we present case studies in development of reliable methods based on mass spectrometry (MS) to characterize a protein–hapten drug substance during late-phase process validation. This protein is…

Providing Lipids Boosts Protein Productivity: Testing a Feed Supplement with Multiple Cell Clones and Media Formulations

As the biologics (and now biosimilar) markets continue to grow, pressure increases on biomanufacturers to reduce cost of goods sold (CoGS). One way they can reduce cost is by increasing protein productivity in terms of protein titer per volume of culture. Media optimization is a key strategy for increasing protein productivity. In the past few decades, average titers across the industry have increased greatly — from <0.5 g/L in the 1980s to >3 g/L today, and it is not uncommon…