Separation/Purification

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…

Sticking In or Standing Out? Dichotomy in Vaccine Purification By Chromatography

A general vaccine purification strategy can be divided into three stages, with one or more steps for each stage. The first stage is to concentrate and isolate the target molecule quickly to remove it from conditions that could lead to its inactivation or loss. Intermediate purification seeks to remove remaining contaminants, typically using an orthogonal approach. That is followed by a polishing step in which trace impurities are removed through high-efficiency steps because those impurities usually are similar to the…

Process Analytics and Intermediate Purification of Bispecific Antibodies with a Non-Affinity Platform

The therapeutic benefits of monoclonal antibodies (MAbs) have been demonstrated in recent decades with uncontestable success as treatments for human disease. Despite MAbs’ key features such as specificity, selectivity, and safety, the format has limitations (1, 2). Bispecific antibodies may overcome number of difficulties (3). Multiple formats of bispecific antibodies have been developed, although only the κλ-body is fully human and devoid of linkers or mutations. It requires no genetic modifications of heavy and light chains and results in bispecific antibodies…

Virus Segregation During Purification Processes: Calculation of Critical Potential Carryover of Viruses

Before a pharmaceutical product is introduced into humans, either in a clinical trial or as a marketed product, virus safety must be evaluated carefully. Virus safety normally is ensured using a three step complementary approach: selecting and testing cell lines and/or raw materials for the absence of viruses, testing the product at appropriate steps of production, and assessing the capacity of a production process to clear infectious viruses (1). The latter (also referred to as viral clearance) is the subject herein. Spiking studies are conducted to evaluate the capacity of a 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…