Downstream Validation

CMC Strategy Forum Special Focus Series: Part 2 Product-Related Impurities, An Overview

Introduction by Cheryl Scott 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. This process is meant to help ensure that biopharmaceutical products manufactured with advancing technologies in a regulated environment…

Detecting the Broad Spectrum of Pyrogens with the Human Whole-Blood Monocyte Activation Test

In the early 20th century, some patients injected with the drug Salvarsan experienced febrile reactions due to contamination of the drug’s distilled water. That incident (involving the first effective treatment for syphilis) prompted not only the widespread use of injectable drugs, but also the need for pyrogen control. Pyrogens constitute a heterogeneous group of microbial and nonmicrobial substances that include those derived from Gram-negative and Gram-positive bacteria such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively, as well as particles…

Defining Your Product Profile and Maintaining Control Over It | A Look Back with Emily Shacter

This is a transcript from a Q&A interview with Emily Shacter, PhD, Consultant, ThinkFDA LLC (former FDA Scientist and Regulator). We will be talking today about the CMC Forum that was published back in 2005. We are revisiting it in the magazine to specifically update our understanding of how to maintain process control; understanding your process. In general, how do you feel the discussions in the four-part paper from 2005 has held up after 10 years? Emily: I think they…

Identification and Quantification of Heat-Shock Protein 70: A Major Host-Cell Protein Contaminant from HEK Host Cells

Recombinant therapeutic proteins are commonly produced by cell lines such as Chinese hamster ovary (CHO), human embryonic kidney (HEK) 293, murine myeloma (NS0), and Escherichia coli bacterial cells. Host-cell proteins (HCPs) are indigenous proteins produced by those expression hosts and considered to be process-related impurities generated from the cell culture process (1). HCPs are potentially harmful and immunogenic to patients, and they can compromise the stability of protein drugs (2–4). For those reasons, HCPs must be consistently removed or reduced…

Evaluation of a Variable-Pathlength Spectrophotometer: A Comparable Instrument for Determining Protein Concentration

Protein concentrations in bioprocessing are determined by multiplying the measured absorbance of UV light as it passes through a sample by the protein extinction coefficient. Conventional spectrophotometer measurements are based on a fixed pathlength depending on the cuvette used to hold the sample (typically 10 mm). Only a small portion of the UV curve is linear at that pathlength. As a result, conventional spectrophotometers have a limited linear range and are unable to measure a large range of protein concentrations…

Fundamental Strategies for Viral Clearance Part 2: Technical Approaches

Viral safety is required for biologics manufactured to treat human diseases. Although significant improvements in ensuring viral safety have been made over the past few decades, “zero risk” of viral contamination is a myth. Viral contamination risk can be carefully managed by screening raw materials, testing process intermediates, and evaluating how effectively manufacturing processes remove and inactivate viruses. Viral clearance studies verify virus removal or inactivation by a manufacturing process. Although regulatory agencies have expectations for the designs of those…

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

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…

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

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…

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

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…

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

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…