Downstream Processing

Predicting Viral Clearance in Downstream Process Development

As viruses can arise during the manufacture of biopharmaceuticals, regulatory agencies require viral clearance validation studies for each biopharmaceutical prior to approval. These studies are typically conducted in biosafety level (BSL)-2 facilities and require large capital and human resources. The use of an accurate, economical, and quantifiable noninfectious viral surrogate would enable downstream purification scientists to study viral clearance throughout process development. This report explores the use of a BSL-1 compatible, noninfectious MVM particles to predict viral clearance results over…

eBook: Chromatography Resins — Addressing Challenges in Biologic Purification Workflows

Although chromatography remains the backbone of downstream workflows, selecting appropriate technologies to optimize processes can be challenging. Numerous resin options are available, and the fact that most biologics are large, complex, and inherently unstable further complicates development of a robust workflow. Because chromatography processes can alter a biologic in ways that could impair its intended therapeutic function, investment in process development is critical. Techniques such as design of experiments (DoE) can be used to identify the best approach during process…

Viral Clearance in a Downstream AAV Process: Case Study Using a Model Virus Panel and a Noninfectious Surrogate

Over the past decade, adenoassociated virus (AAV) vectors have become established as leading gene-delivery vehicles. In 2017, the pipeline for gene therapies included 351 drugs in clinical trials and 316 in preclinical development (1–4). As those candidates advance, significant efforts are being made in process development and manufacturing for viral vectors, with the overall goal of reducing process impurities while maintaining the highest possible process yield. To address that goal, industry suppliers have developed innovative AAV-specific separation technologies. Thermo Fisher…

eBook: Buffers — Navigating New Demands on Downstream Raw Materials

Bioreactor titers for monoclonal antibody (MAb) processes have increased significantly since the dawn of the biopharmaceutical industry, yet such gains have instigated bottlenecks for critical high-volume raw materials used in downstream processing, such as buffer solutions. As downstream purification is required for most, if not all, biopharmaceutical products, buffers and their preparation are topics that concern nearly every drug company. But those topics rarely receive direct attention. This BPI eBook explores what factors prompted the current buffer bottleneck and what…

How to Improve the Capturing of Antibody Fragments

Some of the latest promising biopharmaceutical drug substances are antibody fragments. Antibody fragments are either separate functional subunits of antibodies or recombinant molecules, which, just like antibodies, are composed of immunoglobulin domains. These drugs offer several therapeutic advantages over conventional monoclonal antibodies. Upstream processing for antibody fragments is easier than it is for standard antibodies. Recombinant-based antibody fragments can be modified to meet specific needs of affinity, avidity, valence, and action mode. They also can be produced in prokaryotic cells…

Removing Oligomers of a Recombinant Human Therapeutic Hormone:
Evaluation of Chromatographic Options for Effectiveness

Aggregation is a common cause of protein instability, which renders a biologic product unfit for therapeutic use. Sometimes it is difficult to purify monomeric proteins from oligomers because of similarities in their isoelectric points (pIs). Proteins such as hormones have pI ranges similar to their oligomers and thus can be difficult to separate out using a conventional polishing chromatographic step such as ion exchange. With those pI similarities, removal of oligomers to a considerable extent by ion exchangers can compromise…

Evaluation of a Novel Peptide-Based Affinity Ligand for Human IgM Purification: Use of an Automated Liquid-Handling System for Rapid Assessment of Binding Kinetics and Capacity

One-step affinity purification of antibodies is a powerful and widely used tool in the biopharmaceutical industry. Although different strategies can be used to purify immunoglobulin isotype G (IgG), the larger antibody isotype IgM has limited options. Human IgM is the largest antibody and primarily exists as a pentamer in the bloodstream (1). IgM molecules exhibit higher avidity than other antibodies do and serve as essential activators for the complement cascade (1–3). Approaches to IgM purification with hydroxyapatite and ion-exchange (IEX)…

Nontargeted HCP Monitoring in Downstream Process Samples: Combining Micro Pillar Array Columns with Mass Spectrometry

Protein biopharmaceuticals have emerged as important treatments for diseases with otherwise unmet medical needs. These biologics are produced by recombinant mammalian, yeast, or bacterial expression systems. Along with therapeutic proteins, those cells produce endogenous host-cell proteins (HCPs) that can contaminate biopharmaceutical products despite multiple purification steps in downstream processing. Because such process-related impurities can affect product safety and efficacy, they need to be monitored closely. Multicomponent enzyme-lined immunosorbent assays (ELISAs) presently are the workhorse method for HCP testing, with high…

eBook: Ion-Exchange Chromatography for Modern Biopharmaceutical Purification

Understanding the functionalities of chromatography resins can improve the product yield and purity in a biotherapeutic purification workflow. Ion-exchange (IEX) chromatography separates biomolecules on the basis of charge. For several reasons, it is the most widely used separation tool for purification of biopharmaceutical products. IEX is a well-characterized purification method with high binding capacity and flexible selectivity. It also works with mild operating conditions that help to preserve the biological activity of a biopharmaceutical drug substance. That versatility enables several…

Streamlining Industrial Purification of Adeno-Associated Virus

With its first licensed therapeutic now marketed worldwide (1), adeno-associated virus (AAV) has become a preferred vector for gene therapy. However, unlocking its full potential still poses challenges, many of which are associated with purification. The first involves the transition from upstream to downstream processes. AAV-bearing lysates are laden with debris that foul filtration media and limit or prevent concentration. Another challenge involves reduction of soluble host-cell DNA, which is complicated by its strong association with nucleoproteins. A third involves…