Separation/Purification

Streamline Manufacturing of Antibody-Based Therapeutics with Novel Purification Approaches

The global market for antibody therapeutics is rising rapidly as newer, engineered modalities such as bispecific antibodies and antibody fragments are entering clinical studies in record numbers. Protein A affinity chromatography has been widely adopted for the purification of traditional therapeutic monoclonal antibodies (MAbs) by binding to the Fc region. However, with the development of novel engineered antibody formats lacking that region, protein A often underperforms or fails completely. Downstream process scientists are tasked to evaluate and implement alternative solutions…

A Scalable, Two-Step Purification Process for Plasmid DNA

Plasmid DNA (pDNA) plays a critical role in biopharmaceutical manufacturing — e.g., by providing a template for mRNA synthesis, delivering genes of interest to production cell lines and viral vectors, and even serving as a basis for DNA vaccines. Although plasmid production in microbial hosts is a well-characterized process, downstream purification of the needed supercoiled (sc) forms can be time-consuming, resource-intensive, and difficult to scale. The latter concern is especially problematic considering how many applications require pDNA — sometimes large…

Using Automated Immunoassays for HCP Analysis in Early Bioprocess Development

Biopharmaceutical drugs are produced by a number of expression host systems, mainly mammalian (e.g., Chinese hamster ovary, CHO) and microbial (e.g., Escherichia coli) cells. The goal of subsequent purification steps is production of a pure drug substance, which is essentially free of product variants and process-related impurities such as host cell proteins (HCPs) and nucleic acids. Depending on the process, HCPs can represent the total proteome of a host cell line and accordingly are highly diverse in size, charge, hydrophobicity,…

Scalable mRNA Affinity Capture: Supporting Development and Production of mRNA-Based Therapeutics

As a direct result of the COVID-19 pandemic, mRNA-based therapeutics have made significant clinical and commercial progress. With many such therapeutics filling up the clinical pipeline for a growing number of indications, the biopharmaceutical industry is investigating efficient and scalable manufacturing techniques. For example, Thermo Fisher Scientific has developed a novel affinity chromatography resin with a poly(styrene-co-divinylbenzene) backbone and with throughpores to isolate and purify mRNA at various scales. Because the POROS Oligo dT(25) affinity resin binds mRNA through a…

Control of Host Cell Proteins in Monoclonal-Antibody Bioprocessing: Using Proteomic Analysis To Understand Impurity Clearance and Persistence During Purification

Downstream process development can proceed like a detective novel, starting with evidence of something seriously wrong and rapidly evolving into a “whodunit.” The evidence often comes as precipitate particles in what is supposed to be a stable formulation. The whodunit takes the form of root-cause analysis into the degradation mechanism of a biopharmaceutical product or of a key ingredient in its formulation. And the culprit often turns out to be an enzyme present in such small quantities as to be…

Chromatographic Purification: Data Science, Chemistry, and Process Engineering Are Driving Bioprocess Innovation Forward

When people decry “the lack of innovation” in biomanufacturing, I often find myself scratching my head. Maybe we’re working from different angles on the concept — for some, it might mean only those advances that are truly disruptive and replace a previous paradigm completely. But what I see in the biopharmaceutical industry currently is an explosion of new ideas, fresh approaches to established technologies, and incremental improvements that all add up to paint a dynamic picture of innovation indeed. “Creativity…

Demystifying Mixed-Mode Chromatography Resins: Emerging Applications for Purification of Non-MAb Protein Therapeutics

Although monoclonal antibodies (MAbs) still represent the most common and most lucrative drug class in the biopharmaceutical industry, enthusiasm is growing for other recombinant-protein products. Clinical pipelines and even commercial portfolios are beginning to feature a breadth of nonantibody proteins and fragments as well as variations on the conventional-antibody theme: e.g., single-chain variable fragments (scFvs), fragment antigen-binding (Fab) products, and single-domain antibody fragments (also called nanobodies). By exploring such formats, drug developers hope to continue leveraging the efficacy advantages of…

Platform Optimization for Efficient AAV Purification: Insights from a CDMO

The number of clinical studies continues to increase for candidate gene therapies based on adenoassociated virus (AAV) vectors. Application of different AAV serotypes has enabled drug developers to target a large panel of tissues and to address a breadth of diseases — but with the drawback that each serotype necessitates a distinctive purification process. Such development work requires considerable resources and results in long process-development timelines, which can cause significant delays to entering clinical trials. Thus, gene-therapy companies and contract…

Intensification of Fab-Fragment Purification: Multicolumn Chromatography Using Prepacked Protein L Columns

Antibody fragments — such as fragment antigen-binding (Fab) domains, single-chain variable fragments (ScFvs), and heavy-chain variable domains (nanobodies) — have emerged as increasingly important therapeutic and diagnostic alternatives to full-length monoclonal antibodies (MAbs) for a multitude of diseases. Whereas MAb downstream processing is well established and easy to scale based on protein A capture, the purification of antibody fragments is just on the verge of standardized processing. The most promising candidate for effective capture of those containing a kappa light…

Purification of Hepatitis B Virus Surface Antigen for Vaccine Products: Impact of Ligand Density on HBsAg Purification By Immunoaffinity Chromatography

According to the World Health Organization (WHO), more than 350 million people worldwide are chronic carriers of hepatitis B virus (HBV) (1). Around 25% of carriers develop liver cirrhosis and/or carcinoma, making HBV responsible for the deaths of one million people annually (1). The virus has a spherical shape with a lipoprotein coating mostly of HBV surface antigen (HBsAg) (2). Knowing that, drug developers have created recombinant HBV vaccines based on HBsAg synthesized in yeast or mammalian cells (3, 4).…