Downstream Processing

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…

Optimization and Scale-Up of HCIC-Based MAb Purification Processes, Part 1

Monoclonal antibodies (MAbs) serve important medical needs in cancer treatment as well as that of autoimmune and infectious diseases (1). Antibodies are also widely used in clinical diagnostic assays. They can be coated on solid surfaces to bind specific analytes, conjugated to reporter molecules (either as whole antibodies or fragments) for analyte detection, used in sensitivity panels for lot-release testing, and supplied as positive controls in diagnostic kits (2). Our study evaluates the use of hydrophobic charge-induction chromatography (HCIC) for…

BioProcess Theater: Formulation, Fill and Finish

Fragile proteins and other biomolecules need protection as stable drug products. The larger a molecule is, the more difficult it will be to make, ship/store, and administer to patients. Biotech drug formulators have many concerns to juggle in their work, beginning with the physicochemical characteristics of an active molecule and including the reliability, cost, and availability of analytical methods; the array of excipients and adjuvants on the market; evolving delivery methods and devices; patient preferences and behavior, as well as…

Cadence™ Single-Pass TFF Coupled with Chromatography Steps Enables Continuous Bioprocessing While Reducing Processing Times and Volumes

Continuous bioprocessing initiatives have accelerated in recent years. The transition from batch to continuous bioprocessing offers numerous advantages, including process time savings and lower capital costs due to smaller equipment, tanks, and tubing sizes. Additionally, hold tanks may no longer be needed between unit operations, which can be especially valuable for facilities with limited manufacturing floor space. Furthermore, reduced system hold-up volumes improve product recoveries and also contribute to the smaller system footprint of continuous bioprocessing systems. One unit of…

Volume Reduction and Process Optimization with Cadence™ Inline Concentrator

Pall’s Cadence™ single-pass tangential flow filtration (SPTFF) technology provides a simple solution for continuous concentration and optimization of Downstream Processing (DSP) steps. It eliminates the conventional TFF recirculation loop and allows product to be concentrated in a single pump pass. The single-pass operation eliminates any mixing or foaming issues and exposes the product to low shear; thus, it is optimal for the processing of fragile and shear-sensitive molecules as well. The single-pass feed flow rates are lower compared to conventional…

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…

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…

Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal

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…

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…

Fundamental Strategies for Viral Clearance – Part 1: Exploring the Regulatory Implications

Over the past several decades, biologics such as monoclonal antibodies (MAbs) and recombinant proteins have provided therapeutic benefits and efficacy for the treatment of human disease. Completion of the human genome project (launched in 1990) produced a draft of the genome in 2001. A full sequence was published on the 50th anniversary (2003) of the initial publication of Watson and Crick’s papers on the double-helical structure of DNA (1). That large volume of genetic information has been translated into usable…