Separation/Purification

Adenovirus Downstream Process Intensification: Implementation of a Membrane Adsorber

Historically, companies developing vaccines have used attenuated pathogens, inactivated infectious agents, or antigenic constituents purified from pathogenic sources. In the past 20 years, technological advances such as recombination and viral vectors, have enabled development of vaccines against diseases with previously no available treatments (1). Viral vectors have become one of the most rapidly evolving and promising fields in vaccinology and regenerative medicine. In addition to preventing infectious disease, they have a broad range of potential applications, including treatment of hereditary…

Rapid Mammalian Cell Harvest Without Centrifugation for Antibody Purification: Using a Novel System for Cell Culture Media Clarification

Monoclonal antibody (MAb) expression systems typically use signal peptides to ensure secretion of antibodies into cell culture media. Although that reduces the complexity of purification and prevents the need for cell disruption, it does require using expensive and time-consuming techniques to separate cells from antibody-containing cell culture fluids. In this study, we describe our tests of the novel Sartoclear Dynamics Lab V system (Sartorius S Lab Instruments GmbH and Co. KG) for rapid clarification of cell culture media without requiring…

Making Downstream Processing Continuous and Robust: A Virtual Roundtable

Current biomanufacturing is driven to pursue continuous processing for cost reduction and increased productivity, especially for monoclonal antibody (MAb) production and manufacturing. Although many technologies are now available and have been implemented in biodevelopment, implementation for large-scale production is still in its infancy. In a lively roundtable discussion at the BPI West conference in Santa Clara, CA (11 March 2019), participants touched on a number of important issues still to be resolved and technologies that are still in need of…

Tangential-Flow Electrophoresis: Investigation of Factors Involved in an Effective Separation of Human Serum Albumin from Human Plasma

Human plasma is a complex mixture of biomolecules such as serum albumin, immunoglobulins, coagulation factors, and others (1). These important protein biomolecules often are present at low levels or lacking in affected patients with certain life-threatening conditions. To extract those valuable proteins, a number of purification methods have been developed over time. Plasma fractionation can be traced back to the middle of the 20th century, when Edwin Cohn of Harvard University developed the first industrial process to purify proteins from…

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…