Author Archives: Pete Gagnon

challenges with exosome therapy

eBook: Challenges in Industrial Process Development of Exosome-Based Therapies: Characterizing and Managing Diversity

The traditional classification of extracellular vesicles (EVs) includes three types: exosomes, microvesicles, and apoptotic vesicles. Each type arises from a distinct origin and exhibits distinct characteristics. The problem is that their size ranges overlap and that the major surface proteins presented by exosomes also are present on the surfaces of microvesicles and apoptotic bodies. This makes it a challenge for process developers to identify the vesicle fraction that best serves a particular exosome therapy. Anion-exchange chromatography (AEC) can fractionate EVs…

CIMmultus PrimaS: The Next-Generation Anion Exchanger for Today’s mRNA-Based Vaccines

CIMmultus PrimaS multimode ligand bioprocessing technology represents a new class of anion exchangers for fast, efficient, one-step purification of single-stranded mRNA at ambient temperature. DNA, dsRNA, and proteins are eliminated with a high-salt wash. Then ssRNA is fractionated by size in a pH gradient that removes short transcripts and fragments. Convective mass transfer and laminar flow through the technology’s monolithic architecture maintain high capacity, high resolution, and low shear stress — even at flow rates >5 column volumes per minute.…

Setting a Cornerstone for Platform Purification of Exosomes

Exosomes are a subject of rapidly growing therapeutic interest in the biopharmaceutical industry for two principal reasons. The first reason is that they are the primary communicators of instructions from source cells to target cells. Exosome surface features define their destination. They recognize complementary features on target cells, dock with them, and deliver their programmed instructions in the form of microRNA. The second reason is that exosomes are immunologically silent. As normal human cell products, and by contrast with gene…

Platform Purification of Clinical-Quality Bacteriophages

Antibiotics are unable to keep pace with infectious diseases, and the use of bacteriophages is a timely solution to that problem. But implementation of bacteriophages involves overcoming some challenges. Broad-spectrum clinical efficacy will require numerous phages. In turn, cost-effective development of purification procedures will require a platform approach in which one basic process can accommodate all species. That includes the ability to reduce endotoxins to appropriately low concentrations despite the high loads that occur in gram-negative production systems. Monolithic chromatography…

Emerging Tools for Exosome Purification and In-Process Monitoring

This eBook introduces new analytical approaches that enable in-line chromatographic detection of exosomes. One approach can discriminate extracellular vesicles from nonvesicle contaminants, and one potentially can discriminate exosomes from other vesicles. Examples illustrate how they enable development of more effective and better documented purification methods. The special qualifications of monolithic chromatography media for exosome purification are discussed. New process tools designed to accommodate some of the special challenges of exosome purification are introduced. Exosomes represent one of several species of…

Intensification of Influenza Virus Purification: From Clarified Harvest to Formulated Product in a Single Shift

Influenza is a global respiratory disease with an estimated mortality of up to a half million people per year (1). The majority of traditional influenza vaccines are still produced in eggs. Downstream processing typically consists of clarification by centrifugation, concentration by ultrafiltration, and purification by ultracentrifugation (2). Recombinant vaccines are most often purified by chromatography. Chromatographic purification of viruses already has achieved major improvements in recovery and scalability (3), but it also is important because it enables virus purification to…

Hydrogen Bond Chromatography – A New Tool for Enhanced Separation of Proteins from Virus Particles and Other Very Large Biologics

CIMmultus ADC monoliths from BIA Separations are the first chromatography products in the field to exploit hydrogen bonding as the primary binding mechanism. These products bring a unique new selectivity to all purification challenges, but they are especially distinctive in their ability to retain large biomolecules more strongly than small ones. They enhance removal of fragments, aggregates, and viruses from proteins, and they enhance removal of proteins and other small contaminants from viruses and other very large biologics. H-Bond ADC…

IgG Purification By Ultrafiltration: Time for Another Look

One of the early disappointments in development of immunoglobulin G (IgG) purification technology was ultrafiltration on membranes with 50–100 kDa cutoffs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that most host cell proteins were smaller than that. IgG was retained. Parallel concentration and buffer exchange could be performed going into a follow-on polishing step. These features made it an obvious candidate for initial capture, but it did not perform as hoped. Membrane fouling sabotaged its concentration–diafiltration potential, and prohibitive…

Recent Advances in Endotoxin Removal: An Upgrade to a Traditional Method and a New Adsorption Chemistry

Endotoxin contamination has been the bane of the bioprocessing industry since its inception. Endotoxins are everywhere: They are toxic and/or interfere with every type of therapeutic, diagnostic, and research product; they are indestructible within the limits of product tolerance; and they are difficult to remove (1–4). Beyond that, they interact with various biological species in ways that prevent accurate measurement (5, 6). Managing these issues has been a focus of the industry for at least half a century, yet it…

The Secret Life of Protein A

Affinity chromatography with protein A has become the foundation for purification of nearly every therapeutic IgG in commercial production. One of the features most responsible for its success has been its compelling simplicity. IgG binds. Contaminants do not. Load, wash, and elute pure IgG. In the real world, however, protein A does not elute pure IgG. It typically contains several hundred to a few thousand parts per million (ppm) contamination by host-cell proteins (HCPs) and other contaminants. Numerous studies demonstrate…