Downstream Processing

The Multi-Mode Mimetic Ligand Library: A New Tool for Rapid Development of Downstream Processes

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Recent developments in downstream processing of biomolecules — including continuous processing, bind–elute affinity capture, and flow-through polishing steps — have increased the need for greater selectivity from chromatography adsorbents. This has led to the introduction of a new generation of adsorbents: so-called “mixed-mode” or multimodal ligands. They provide greater selectivity and tolerance to process buffer composition than either ionexchange chromatography (IEC) or hydrophobic-interaction chromatography (HIC) alone can provide. Learn more in this Special Report from Steve Burton, Chief Executive Officer…

April 20, 2018

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

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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…

April 20, 2018

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

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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…

April 19, 2018

Filter-Based Clarification of Viral Vaccines and Vectors

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Viral vaccines rely on the antigen properties of a virus or virus-like entity to trigger an immune response and induce immune protection against a forthcoming viral infection. Through development of recombinant viral vaccines, developers can reduce risks associated with the presence of live and inactivated viruses. Instead, recombinant vaccines induce immunity against a pathogen by relying on the capacity of one or more antigens delivered by means of viral vectors or the baculovirus/plasmid system (1). Viral vaccines are formulated with…

April 19, 2018

A Two-Step Purification Process: Application of HIC Membrane Chromatography in a Disposable 2,000-L Clinical Facility

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Given paradigm shifts in the biopharmaceutical industry over the past decade, product development timelines are squeezed as the number of molecules entering clinical development continues to increase. Manufacturing facilities, especially those supplying clinical trial materials, have had to adapt to this trend. One popular approach is to have fully disposable equipment that allows for quick product changeover and flexible manufacturing capacity to respond to variable clinical demand. Although many facilities-related technologies exist to support that concept (e.g., disposable probes and…

March 23, 2018

IgG Purification By Ultrafiltration: Time for Another Look

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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…

March 23, 2018

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

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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.…

March 23, 2018

Advanced Viral Clearance Study Design: A Total Viral Challenge Approach to Virus Filtration

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Biologics derived from mammalian organisms have been accepted for therapeutic use for almost a century (1). However, these pharmaceuticals have the potential for contamination with pathogenic adventitious agents such as viruses. With cell-line–derived recombinant proteins, the viral risks commonly include viruses in the Retroviridae and Parvoviridae families (2). As patient safety and manufacturing facility suitability became significant concerns in the 1980s and 1990s, several industry and regulatory bodies reached consensus on how to approach the unique challenges of viral safety…

March 23, 2018

Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal

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Most recombinant monoclonal antibodies (MAbs) are produced by mammalian cells. Because biopharmaceuticals derived from mammalian tissue culture carry the risk of adventitious virus contamination, regulatory agencies expect risk-mitigation strategies to include validation of purification unit operations for their ability to clear viruses (1). Guidelines from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) describe how to prove viral clearance in downstream purification processes using an orthogonal approach (2). Viral log10 reduction values (LRVs) are…

March 23, 2018

New Dimensions in Single-Use Filtration

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Whether viral vectors are clarified or the bioburden after cell harvest needs to be reduced to recover antibodies, such applications in biopharmaceutical production require large filtration areas. Single-use technologies are indispensable in many such bioprocesses. Although some single-use filter assemblies have reached their limits, Sartorius Stedim Biotech has made developments to revolutionize these production steps. Scale-Up Limitations in Single-Use technology Conventional stainless steel process systems have been established for decades in the pharmaceutical industry. They are the basis of safe…

February 14, 2018