Assays

Amplifying the Possibilities

Polymerases are natural enzymes that are vital to nucleic acid synthesis: DNA polymerase for replication of deoxyribonucleic acid and RNA polymerase for replication of ribonucleic acid. Thus all living things make and use polymerases of their own. But in 1969, the University of Wisconsin’s Thomas D. Brock and Hudson Freeze identified a new species of extremophilic bacterium thriving at 160 °F (70 °C) in a hot spring in Yellowstone National Park. In time, heat-tolerant polymerase isolated from Thermus aquaticus (Taq)…

Antibodies, Bioassays, and Cells

It’s no surprise that immunochemistry forms a broad and solid basis of biopharmaceutical analytical laboratory work. Immunochemicals include antibiotics and antigens, nucleic acids and nucleotides, enzymes, lipids, antioxidants, probes and dyes, and proteins and peptides. Available from companies such as Advanced Immunochemical, Immundiagnostik, Lampire Biological Laboratories, and Rockland Antibodies and Assays, their many uses include antibody isotyping and fragmentation. Adjuvants, buffers, assay kits, target biomolecules, and phage-display systems support those applications. Because background and off-target effects complicate the study of…

Rapid Detection of Pandemics

A Coronavirus — like severe acute respiratory syndrome (SARS) — is back in the headlines. On returning from a trip to Saudi Arabia in summer 2012, a Qatari national was struck down by a mystery respiratory illness. Because of inadequate diagnostic capabilities, the patient was transferred from Qatar to London for intensive-care treatment and diagnosis. The UK Health Protection Agency (HPA) confirmed infection with the same Coronavirus strain discovered by a Dutch team following the death of a Saudi national…

A Host Cell Protein Assay for Biologics Expressed in Plants

    Host cell proteins (HCPs) can be present in significant amounts in biological products through copurification with a recombinant protein-drug substance. Purified active ingredients developed for human use must be free of all contaminants — including residual HCPs — to minimize the incidence of immunogenicity against a drug product or its trace contaminants (1).   HCP assays allow for monitoring host-related impurities during product development and process development. Most are in the enzyme-linked immunosorbent assay (ELISA) format for detecting…

Balancing the Statistical Tightrope

During one development meeting early in my industrial career, a process development group member asked me whether the value my group had reported in one result was okay to use. I confidently replied “Yes, it’s fine. It’s about 40, somewhere between 38 and 42. The other person raised his eyebrows. “About 40?” In response, I somewhat awkwardly mumbled “Yes, probably…about that” — an answer not met with full understanding, but rather concern. My answer hadn’t been incorrect. The result was…

Development of an In-House, Process-Specific ELISA for Detecting HCP in a Therapeutic Antibody, Part 2

    During biopharmaceutical manufacturing, final drug products can get contaminated with host-cell proteins (HCPs) derived from a production cell line. HCPs can elicit adverse immune responses, so regulatory authorities require accurate monitoring of their presence and concentration in final drug products. Because they are robust and offer good throughput, enzyme-linked immunosorbent assays (ELISAs) are the first choice for HCP detection to monitor product quality. Generic ELISA kits are commercially available for HCP detection with a number of commonly used…

Development of an In-House, Process-Specific ELISA for Detecting HCP in a Therapeutic Antibody, Part 1

    After production and purification of biopharmaceuticals generated by cell culture expression systems, endogenous cell line proteins — commonly referred to as host-cell proteins (HCPs) — sometimes contaminate finished products. HCPs can elicit an immune response following administration of those drugs to patients (1), and cause potentially deleterious side effects. It is therefore imperative to minimize HCP contamination in finished biologics. Regulatory health authorities require monitoring of HCP contamination. They expect validation of each purification process to demonstrate its…

A Biomass Monitor for Disposable Bioreactors

    Of the available on-line biomass assay types, radio-frequency impedance spectroscopy (RFI, often referred to as capacitance) is generally regarded as the most robust and reliable method for monitoring viable biomass during fermentation and cell culture. The first article to show that capacitance could be used to estimate microbial biomass dates back over 20 years (1). Today the technology is routinely used for monitoring and controlling mammalian cell culture processes and high-density yeast and bacterial fermentations in research, process…

Measuring Manufacturing Cost and Its Impact on Organizations

    The first article in this periodic series reviewed the impact of cost pressures on the biopharmaceutical industry, in particular the challenges the industry faces in relation to high capital costs, complex processes, and long product development cycles (1). Here we examine what companies are doing to assess costs in decisions about process and technology choices relating to manufacturing of biologic drug substances. We will look into what companies are currently doing and what they need to be doing…

Are Generic HCP Assays Outdated?

    Biomanufacturers face a conflict between low-cost generic host cell protein (HCP) assays and highly sensitive but more costly process-specific HCP assays that are usually not initiated until the proof-of-concept stage. But drug developers cannot expect sufficient sensitivity from most commercially available generic assays. For some companies, multiproduct HCP assays could offer a solution to the dilemma. Biopharmaceutical manufacture using genetically modified microorganisms and cell lines is typically associated with contamination by process-related impurities. One of the most important…