Downstream Validation

Reagent Clearance Capability of Protein A Chromatography: A Platform Strategy for Elimination of Process Reagent Clearance Testing

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During the manufacturing of monoclonal antibody (MAb) products, many process reagents are used for cell culture and MAb purification to facilitate and control process performance. Process reagents are considered to be process-related impurities, so demonstration of their clearance is required for the chemistry, manufacturing, and controls (CMC) information submission of an investigational new drug (IND) application (1, 2). These reagents may be classified into two categories: generally recognized as safe (GRAS) reagents and potential safety concern (PSC) reagents (3). GRAS…

May 12, 2015

The Potential Application of Real‑Time Release Testing for the Biomanufacture of Autologous Cell‑Based Immunotherapies

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Cell-based immunotherapies (iTx) are emerging as a truly transformative therapeutic modality that is both complementary and convergent with existing regenerative medicine approaches, including gene therapy, cell therapy, and tissue engineering (Figure 1). Critically, iTx offer step-change improvements in efficacy compared with current standards of care (1) for a range of clinical indications and unmet therapeutic needs — particularly oncology. The clear efficacy of iTx is in contrast with some previous regenerative medicine approaches, including early mesenchymal stem cell (MSC) therapies…

April 14, 2015

Hamster Phospholipase B-Like 2 (PLBL2): A Host-Cell Protein Impurity in Therapeutic Monoclonal Antibodies Derived from Chinese Hamster Ovary Cells

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All recombinant protein biotherapeutics must be tested for the presence of residual host-cell protein (HCP) impurities (1–3). The most common analytical method for doing so is a polyclonal sandwich immunoassay. Polyclonal anti-HCP antibodies are selected to recognize the broadest population of HCPs possible. The immunogen and analytical standard are produced from a blank-run fermentation that mimics the production run but lacks the specific biotherapeutic protein. Because of the large number of impurities present in harvested cell-culture fluid (HCCF) that might…

April 14, 2015

Improved Fluorescent Labeling Efficiency of N-Linked, High-Mannose Oligosaccharides: Using 8-Aminopyrene-1,3,6-Trisulfonic Acid (APTS) for Analysis of Glycoproteins

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Glycosylation of proteins, including monoclonal antibodies (MAbs), is recognized as important for the efficacy, immunogenicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of biotherapeutics (1–6). So research and development of protein candidates is increasingly focused on the effects of glycosylation and how its pathway is affected in the Golgi system of cells involved in biosynthetic processes (7). Such attention on glycosylation has helped advance analytical technologies such as high-pH anion-exchange chromatography (HPAEC) (8); normal-phase chromatography (NP- HPLC), hydrophilic-interaction chromatography…

March 10, 2015

Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal

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To date, the majority of recombinant monoclonal antibodies (MAbs) have been produced by mammalian cells. During such production processes, the potential risk of entrained viruses must be critically considered (1). Contamination can arise from animal cell lines or from adventitious viruses introduced during manufacturing. To ensure the viral safety of biotechnology products, companies can take four complementary approaches (2, 3): Using animal-component–free raw materials wherever possible Virus testing of master cell banks Virus testing of unprocessed harvest Performing downscale virus…

February 6, 2015

Unwanted Immunogenicity: From Risk Assessment to Risk Management

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Although vaccines and immunotherapies are designed to engage the human immune system in fighting disease, unwanted immunogenicity can be a major problem for protein-based therapeutics. Some patients produce antidrug antibodies (ADAs), which might lead to drug inactivation or adverse effects. Even human and humanized proteins have proven to be surprisingly immunogenic in some cases, suggesting that immune tolerance requires careful consideration in biologic product design. In rushing to deliver new drugs to market, some biotherapeutics developers have overlooked factors that…

November 10, 2014

Understanding and Controlling Sources of Process Variation: Risks to Achieving Product Critical Quality Attributes

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Biopharmaceuticals include recombinant proteins, vaccines, gene therapies, and drug products derived from stem cell technology. One key characteristic of shared by all biologics is that they tend to be extremely large molecules with complex three-dimensional structures, critical to their functionality. For example, monoclonal antibodies (MAbs) are composed of more than one thousand times larger than a molecule of aspirin (one analogy compares the complexity of a MAb to that of an F16 jet, and the complexity of aspirin to that…

October 16, 2014

Replacing Reverse-Phase Chromatography for Mass Spectrometry: Is Salt-Free Size-Exclusion Chromatography Ready?

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Protein mass is often determined using ultraperformance liquid chromatography (UPLC) coupled with electrospray-ionization mass spectrometry (UPLC/ ESI MS or simply LC-MS). A UPLC system equipped with an ultraviolet (UV) detector serves as an assisting vehicle to deliver purified and separated protein molecules to the mass analyzer. Reserved-phase chromatography (RPC) is the most common chemistry chosen to serve this purpose. For sample purification, not only does RP-UPLC use salt-free mobile phases that are amenable to MS, but it also can efficiently…

October 16, 2014

Preuse, Poststerilization Filter Integrity Testing for Single-Use and Stainless-Steel Installations

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According to current European Union good manufacturing practice (EU GMP), integrity testing of sterilizing-grade product filters should be performed preuse poststerilization (PUPSIT) and immediately after use. In addition, PDA’s Technical Report 26 states that preuse integrity tests are preferably performed after filter sterilization. Performing an integrity test of an already sterilized product filter in-line requires wetting the filter while maintaining the downstream side sterile. The test gas must also be evacuted on the downstream side throughout testing maintaining sterility. The…

October 16, 2014
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UV-Vis Based Determination of Protein Concentration: Validating and Implementing Slope Measurements Using Variable Pathlength Technology

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No longer are scientists bound to the time-consuming, error-prone use of dilution factors and fixed-pathlength measurements in determining the concentration of an analyte in solution. Using the slope spectroscopy technique, the Solo VPE system (from C Technologies) offers a new method of determining analyte concentration based on the Beer–Lambert law and slope derived from absorbance measurements made at multiple pathlengths (1). Mathematics: The Beer–Lambert law is expressed as A = αlc, where A is the measured absorbance, α is the…

September 11, 2014