MAb

Characterization of Postcapture Impurity Removal Across an Adsorptive Depth Filter

In the manufacture of monoclonal antibodies (MAbs), the first purification step following harvest clarification is normally protein A affinity chromatography because of its high selectivity for IgG and high process yield (1, 2). At this stage, a MAb is eluted from a protein A ligand at low pH and then held or adjusted to a low pH (pH ≤ 3.8) for a given amount of time before pH adjustment, usually ≥30 minutes, in a virus inactivation (VI) step targeted at…

A Multidisciplinary Approach to Manufacturing Biotherapeutics

Optimizing antibody manufacturing processes has gone beyond the first-order goal of achieving elevated protein titers and now also focuses on understanding biologic and manufacturing process variables that define cellular machinery and protein quality. A holistic approach to biotherapeutic manufacturing incorporates several applied disciplines such as biology, engineering, process control, signal processing, and modeling to reduce the “black-box” model of cell- based protein production into an operational design space. This is in line with the US Food and Drug Administration’s quality…

Affinity Capture of F(ab’)2 Fragments: Using Twin-Column Countercurrent Chromatography

Antibody fragments are potent active drug substances (1–4). Because they lack glycosylation, they can be produced using different biological expression systems, including yeast and microbial systems as well as mammalian cells. These molecules are interesting as biopharmaceuticals because they are smaller than full-size antibodies and therefore may penetrate better into different tissues. Antibody fragments are cleared faster in biological systems because they lack the Fc antibody structural region (4). However, fragments may be conjugated to increase their size for improved…

Protein A Intermediate Wash Strategies

Protein A affinity chromatography offers efficient monoclonal antibody (MAb) purification and is used extensively in large-scale MAb production. As is the case with most chromatography media, protein A resins often have some degree of nonspecific binding, which causes host-cell proteins (HCPs) to coelute with a MAb. To reduce nonspecific binding interactions, an intermediate wash step can be performed before product elution. Doing so can improve product purity, extend column lifetime, and potentially eliminate a subsequent polishing step. For large- scale…

Immunoglobulin Fc-Fusion Proteins Part 2: Therapeutic Uses and Clinical Development

The potential therapeutic value of many proteins — including enzymes, receptors, cytokines, blood factors and peptides — can be realized by fusing them to the Fc region of human immunoglobulin G. Of the 46 monoclonal antibody (MAb) and MAb-derivative products approved by the FDA to date as human therapeutics, 10 are Fc-fusion proteins (Table 2). Among approved products, several structural variations are represented (Figure 4). In BPI’s October 2014 issue, Part 1 of this review examined the structure and manufacturing…

Immunoglobulin Fc-Fusion Proteins Part 1: Their Design and Manufacture

Over the past three decades, 45 monoclonal antibody (MAbs) and MAb-derivative products have been approved for therapeutic use in the United States (Table 1). One class of antibody derivatives is growing in importance: Fc-fusion proteins. Many biologically active proteins, including receptor ECDs (see “Abbreviations” box), cytokines, enzymes, and bioactive peptides have very short serum half lives because rapid renal clearance limits their exposure in target tissue (and, consequently, their pharmacological effect). The primary reason for fusing a biologically active protein…

Antibody Drug Conjugates: The State of the Art

Monoclonal antibodies have dominated the biopharmaceutical market for over two decades. Few people doubt that their future success in fields such as oncology, inflammation, and autoimmune diseases owes much to the development of antibodies conjugated to cytotoxic drugs. Like all great innovations, this is a breathtakingly simple concept: Combine the targeting specificity of an antibody with a small-molecule drug as an effector component joined to that antibody by means of a small chemical or peptide linker, and you have a…

Bioprocessing Challenges of Antibody–Drug Conjugates

Development of highly potent active pharmaceutical ingredients (HPAPIs) is clearly a pharmaceutical industry trend. Highly potent drug products involve active agents and APIs that are so potent therapeutically (or simply just outright toxic) even in small dose that special precautions are required during their manufacture — particularly when handling the active agents. Such requirements include maximal containment and isolation of the process stream. Worker exposure and environmental release clearly pose problems. The necessity and intensity of containment efforts with HPAPIs…

The Next Step in Homogenous Bioconjugate Development: Optimizing Payload Placement and Conjugate Composition

[Audio Recording] Bringing a new biologic drug to market is a long and expensive process, with research and development (R&D) cycles that can span up to 15 years and may cost over a billion dollars. Biologic drug development also involves significantly more complex manufacturing and CMC components than does development of small molecules. Nonetheless, the pharmaceutical industry is increasingly shifting its R&D efforts to focus on biologic drugs. According to a recent report from Tufts Center for Study of Drug…

Bioconjugation Reaction Engineering and Kinetics Simulation

Bioconjugates represent an important and growing class of pharmaceuticals that include PEGylated proteins, vaccines, and antibody-drug conjugates (ADCs) (1–8). Numerous protein conjugation techniques exist (9). Among the more important conjugation chemistries used for protein therapeutics are N-hydroxysuccinimide (NHS), aldehyde, and maleimide (10–13). To date, process development of industrial biopharmaceutical conjugation reactions has largely been empirical in nature. Typically, many experiments testing different reaction parameters are required to identify optimal process conditions. In some instances, nonmechanistic statistical models can be used,…