Assays

eBook: Bioassays for Biopharmaceuticals: Finding Best Practices in a Quality Systems World

Bioassays are complex and challenging experiments to run reliably with accurate and dependable results. Consistent performance requires a controlled environment and qualified reagents; skilled analysts who understand cell physiology, regulatory requirements, and the latest techniques; and assay protocols that are intelligently developed, characterized, and validated. Here, BPI’s senior technical editor discusses bioassay best practices with representatives of the Biopharmaceutical Emerging Best Practices Association (BEBPA) organization. Topics span quality by design, assay validation, cell banking, potency testing and host-cell protein monitoring,…

Creating Novel Cell Lines By Genome Editing: Simplifying Cell-Based Assays and Improving Production of Biomolecules

Cultured cell lines have a diverse range of applications. They are used broadly by cell biologists, clinicians, tissue engineers, biotechnology scientists, and bioengineers. The most important uses of cell culture are in the cell-based assays and production of biologically active recombinant proteins. In recent years, genome editing has been used widely to study the structure, function, and localization of endogenous proteins in cultured cells. However, applying the same genome editing techniques to cell lines also could improve the propagation of…

Biopharmaceutical Characterization,
Part 1: Biological Assays —
A Conference Report

In late October 2018, KNect365 brought together more than 250 analytical specialists to discuss characterization of well-characterized biologics in Rockville, MD. Speakers from the US Food and Drug Administration joined experts from leading biopharmaceutical companies, service providers, and consultancies, including BPI editorial advisor Nadine Ritter (president and analytical advisor of Global Biotech Experts). She began the final day moderating a special town-hall session where audience members could pose their regulatory questions to a panel of FDA reviewers, and she ended…

A Novel 3D Culture System for High-Throughput Hepatoxicity Screening

Cells grown as three-dimensional (3D) spheroids are thought to more closely mimic in vivo physiology in terms of morphology, structural complexity, and phenotype. Being more physiologically relevant, 3D cultures can be highly predictive for compound profiling and evaluating cytotoxicity, a critical step in evaluating chemotherapeutic drug candidates. Unfortunately, evaluation of drug cytotoxicity traditionally has relied on the use of two-dimensional (2D) cell culture monolayers. When grown in monolayers, cells are not exposed to soluble gradients, are forced into an apical-basal…

Certain Approaches to Understanding Sources of Bioassay Variability

During lifecycle development of a biological assay (bioassay), identifying and reducing sources of variability might be required to improve method performance. Here I recommend some statistical and graphical approaches (consistent with USP <1033>) for practitioners to identify variation from experimental results (1). Sources of Variation in a Bioassay To correctly identify sources of variation in a bioassay, analysts must consider how that bioassay is to be executed. In particular, the experience and technical expertise of each analyst expected to execute…

Statistical Assessments of Bioassay Validation Acceptance Criteria

Analytical linearity as well as assessments of precision and accuracy determine the range for a bioassay (1). USP <1033> recommends comparing confidence intervals (CIs) against target validation acceptance criteria in a bioassay validation exercise, but there are no clear guidelines for determining the criteria (2). Here I address several aspects of a bioassay validation, namely • Linearity (coefficient of determination (R2), slope, and intercept parameters) • Accuracy (%relative bias, %RB) • Precision (percent coefficient of variation, %CV) CIs for the…

The Relationship Between R2 and Precision in Bioassay Validation

Analytical linearity along with assessments of precision and accuracy determine the range for bioassays (1). Practitioners can include coefficient of determination (R2) criteria from a linearity study in the bioassay validation protocol. Herein I illustrate the relationship of R2 to study design and analytical method variation. Overview of the Simple Linear Regression Model Dilutional linearity assesses the “ability (within a given range) of a bioassay to obtain measured relative potencies that are directly proportional to the true relative potency of the…

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…

Development of a Host-Cell Protein Platform Assay for a Chinese Hamster Ovary Cell Line

The Chinese hamster ovary (CHO) cell line is the most prevalent biopharmaceutical expression system and has been proven safe for commercial production of protein therapeutics (1). However, even after multiple purification steps, biopharmaceuticals contain residual host-cell protein (HCP) impurities that pose a potential safety risk to patients (2). Health authorities demand close monitoring of HCP impurities and require sensitive analytical methods with high coverage: the ability to detect a broad range of HCP impurities (3, 4). Polyclonal sandwich immunoassays are…

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…