Separation/Purification

Scale-Up of Twin-Column Periodic Countercurrent Chromatography for MAb Purification

Periodic countercurrent (PCC) processes increasingly are being evaluated as alternatives to single-column batch capture processes. Some of the advantages of PCC processes over single-column processes include shortening of processing time and/or reduction of required resin volume through increased productivity; reduction in resin costs through improved resin capacity use; and reduction in buffer consumption through increased column loading. Those advantages, however, come with increased equipment complexity and hardware costs. PCC processes and systems with two to up 16 columns of the…

Continuous Solids-Discharging Centrifugation: A Solution to the Challenges of Clarifying High–Cell-Density Mammalian Cell Cultures

Clarification is typically the first unit operation in the purification of monoclonal antibodies (MAbs) and other proteins from mammalian cell cultures. This process removes cells and cellular debris from the culture fluid to produce a clarified cell culture supernatant that will be suitable for further purification (1). Before 2000, depth and tangential-flow microfiltration were standard clarification technologies in the biopharmaceutical industry. Process-scale centrifugation was considered to be a significant capital investment, and bioprocessors had limited ability to control (minimizing) the…

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…

Large-Scale Purification of Factor-IX: Comparing Two Affinity Chromatography Resins

Human clotting factor-IX (F-IX) is a glycoprotein that is essential for normal hemostasis (1). A deficiency of F-IX in human plasma is caused by an absence or functional mutation of the F-IX gene that expresses inactive F-IX in plasma. That leads to hemophilia B (“Christmas disease,” named after its first identified patient), a genetic disorder in which the blood-clotting cascade is disturbed (2, 3). The structure and amino-acid sequence of F-IX are similar to those found in other vitamin-K–dependent glycoproteins.…

Addressing the Challenge of Complex Buffer Management: An In-Line Conditioning Collaboration

Preparation and storage of buffers is a challenge for biopharmaceutical companies developing protein-based pharmaceuticals. The need for volumes of buffer to purify increasing upstream titers have become a major bottleneck in biopharmaceutical downstream processing. Italian biopharmaceutical company Kedrion Biopharma collects and fractionates blood plasma to produce plasma-derived therapeutic products for treating and preventing serious diseases, disorders, and conditions such as hemophilia and immune-system deficiencies. To expand its offerings and include the immunoglobulin G fractionate of blood plasma (IgG, an antibody…

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…

Is Continuous Downstream Processing Becoming a Reality?

Over the past 30 years, several biopharmaceuticals have been produced by continuous cell culture processes run in a chemostat or perfusion mode. In most cases, no alternative was available to produce certain unstable molecules (1). However, downstream processing is and has remained a step-by-step batch operation. Continuous processing generally requires more process knowledge, equipment, and technological advances than do batch processes. With the maturity of bioprocessing and increasing awareness of manufacturing costs, companies are focusing on developing continuous downstream processing…

Scalable Purification of Viral Vectors for Gene Therapy: An Appraisal of Downstream Processing Approaches

Gene therapy is the transfer of genetic material to a patient’s cells to achieve a therapeutic effect. Therapeutic DNA is largely delivered using viral vector systems based on adenoviruses (Ad), adenoassociated viruses (AAV), and lentiviruses (LV). With the application of such viral vectors as clinical therapeutics growing, scalable commercial processes (particularly for purification) are being investigated and optimized to best ensure that critical quality attributes (CQAs) are retained. Herein we review viral vector purification techniques and the effect of different…

Downstream Disposables: The Latest Single-Use Solutions for Downstream Processing

Downstream processing has been considered a “bottleneck” in the manufacture of protein biotherapeutics ever since cell culture engineers began dramatically improving production efficiencies around the turn of the century. And as single-use technologies have grown in importance and acceptance, offering more solutions every year, their biggest challenges too have been in the separation, purification, and processing that follows product expression in cell culture. Many of the technologies familiar to process engineers — e.g., centrifugation and chromatography — present technical and…

Single-Use Depth Filters: Application in Clarifying Industrial Cell Cultures

For current process development phases, many biomanufacturers’ attention is directed increasingly to the first unit operation in downstream processing, which is the removal of cells and cell debris from culture broth and clarification of supernatant containing a biopharmaceutical product. Given the high cell densities achievable with both mammalian and microbial cell culture processes, primary recovery can be a significant challenge. The current trend in cell culture is to increase product titers with enriched culture media, improved cell productivity, and increased…