Chromatography

Revisiting Protein A Chromatography

Due to the molecular complexity of monoclonal antibodies (MAbs) and potential impurities in cell culture media before purification (host-cell proteins, DNA, media components) (1), subsequent downstream operations must consistently and reproducibly purify products to ensure safety and efficacy. The latest member of GE Healthcare’s MabSelect family is called MabSelect SuRe LX (2). As Table 1 shows, it has been developed using the same highly cross-linked agarose base matrix and protein A ligand as for other MAb affinity resins (Table 1).…

Understanding Chromatographic Media Ligand Density

    The concept of quality by design (QbD), although not new, has presented implementation opportunities and challenges to both the bioprocessing industry and regulators (1,2,3,4). Tools such as design of experiments (DoE), cause and effect analysis, and multivariate analysis provide for systematic risk assessment and help identify critical quality attributes (CQAs) and critical process parameters (5,6,7). QbD is intended to ensure that manufacturing processes make products that meet predefined quality parameters. Key elements in defining such parameters (quality profile)…

Efficient Aggregate Removal from Impure Pharmaceutical Active Antibodies

Polishing with membrane chromatography (MC) has achieved acceptance as state-of- the-art technology for charged impurities. Traditionally, anion-exchange (AEX) and cation-exchange (CEX) membrane chromatography have been used to remove charged contaminants such as host-cell proteins (HCPs), recombinant DNA, protein A, endotoxins, and viruses. In monoclonal antibody (MAb) processes, polishing steps usually follow a protein A affinity column step. In some cases, CEX capture is applied, either with at least one AEX or a combined AEX and CEX step. The latter may…

Industrial-Scale Biochromatography Columns Address Challenging Purification Needs

    Chromatographic purification remains the most critical step in biopharmaceutical downstream processing. Its purpose is to separate biologic impurities such as host-cell proteins (HCPs), nucleic acids, and oligomers from a target biologic, which must be purified to very high levels (often >99%). Biological separations usually require medium to high salt concentrations and bear inherent risks of microbial contamination in waterbased process streams. Thus they require specifically designed equipment. Depending on process constraints, chromatographic media, and equipment limitations, biochromatographic separations…

Monoliths Open the Door to Key Growth Sectors

    The enabling value of monoliths was strongly in evidence at the 4th International Monolith Symposium, held 29 May – 2 June in the Adriatic resort city of Portoroz, Slovenia. Forty-seven oral presentations and 34 posters highlighted important advances in vaccines, gene therapy, phage therapy for infectious disease, and monoclonal antibodies, as well as continuing advances in the performance of monoliths themselves. As these fields advance in parallel, it becomes increasingly apparent that monoliths offer industrial capabilities substantially beyond…

How to Choose an Industrial Cation Exchanger for IgG Purification

    Cation-exchange chromatography is the third most used industrial method for antibody purification after anion-exchange and protein A affinity chromatography. It is most commonly used as an intermediate step but continues to attract attention as a capture method. This offers obvious cost and cleaning advantages over protein A but also imposes some sacrifices, all of which are discussed in a number of recent articles (1,2,3,4,5). Whichever application may be intended, end users seek a common set of performance characteristics.…

Improving IEX Throughput and Performance with Differentiated Chromatography Sorbents

    Optimized upstream processing and high-productivity cell culture increase not only target protein titers, but also impurity and contaminant concentrations to be removed from large volumes of feedstock. Simultaneously, biopharmaceutical drug production is increasingly driven by manufacturing cost reduction. These facts together increase the pressure on downstream processing and create an urgent need for more productive and streamlined chromatography operations. Key parameters to consider for enhanced process economics in chromatography are higher protein binding capacities at high flow rates…

Novel Affinity Ligands Provide for Highly Selective Primary Capture

    Downstream processing of biopharmaceuticals is costly and time-consuming, often involving multiple steps with significant time and energy expended on maximizing product quality and yield. Affinity chromatography is one of the simplest and most effective methods for purifying protein and peptide therapeutics, offering reduced process steps and therefore higher yields than nonaffinity methods can provide. Protein A is widely used for affinity purification of monoclonal antibodies (MAbs), Fc fragments, and Fc fusion proteins. But it is a challenge to…

Minibodies and Multimodal Chromatography Methods

    Small, genetically engineered immunological constructs are being developed industry-wide for a growing range of in vivo applications. Examples include Fab, F(ab’)2, single-chain (sc) Fv, bis-scFV, diabodies, minibodies, and single-domain antibodies (1). Their small size potentially gives them access to tissues that are poorly accessible by intact antibodies; rapid clearance from blood and nontargeted tissues; lower immunogenic response; and eye-drop, inhalant, or oral administration. We report here on purification of an affinity-matured, humanized, antiprostate stem-cell antigen (PSCA) minibody for…

50 Years of Sephadex Media

It has been 50 years since the first Sephadex paper was published (1). Readers of BioProcess International work in a field that was fundamentally affected by what happened after that paper appeared in 1959. So this anniversary is certainly worthy of a party and a few speeches. But there are lessons to be learned, too. Here we take a look at threads connecting events before and after the discovery of gel filtration chromatography and introduction of the Sephadex product. Interdisciplinary…