Determining the concentration of viruses is a crucial step in any production process. The most commonly used methods for virus quantification are either based on the infectivity of the virus (plaque assay, TCID50) determination of their genomic material (qPCR), or protein content (SRID, ELISA) and are very cumbersome and time consuming. HPLC analytical methods represent a fast alternative to these assays since they provide information on the virus content and purity in a matter of minutes. In this work the…

The interest in continuous bioprocessing to introduce productivity gains and cost savings is rapidly growing. Periodic counter-current chromatography (PCC) is one suitable technological approach, enabling continuous downstream processing. However, implementation in downstream applications is still uncommon mainly due to perceived complexity in operation and process control strategies. This work demonstrates the dynamic control function of ÄKTA™ pcc 75 chromatography system when applied to a protein A capture step operated in a three-column PCC (3C PCC) setup. Dynamic control enables sample…

Purity, recovery, and elution volume are important factors and parameters when screening multiple media (resins) for the development of efficient, robust, and economical production processes. Among a selection of mixed-mode media evaluated for the purification of MAb S, CHT™ Ceramic Hydroxyapatite provided the best monomer recovery at 83% in the smallest elution volume with a target purity of 99.5%. Using a bind and elute strategy provided enhanced purification power compared to flow-through modes.

Strong anion exchange (Q) chromatography has become an industry standard in MAb production. It is a proven technology to remove DNA, viruses, endotoxins and acidic host cell proteins from process feed streams in flowthrough mode. Recent trends show an increasing interest in downstream single-use technologies and flexible biomanufacturing due to advancement in cell culture technology and emergence of biosimilars. Traditional chromatography columns are slow, often oversized and not suitable for flexible biomanufacturing. Conventional membrane adsorbers cannot provide sufficient process robustness…

Host cell protein (HCP) levels in drug products are critical to product quality since HCPs may pose a serious risk to patient safety. The challenge is to accurately quantify the complex mixture of HCP impurities, which vary in properties and abundance depending on the cell line, media, and process parameters. Generic immunoassays commonly used to measure HCP impurities are based on polyclonal antibodies raised against HCPs from non-transfected cell lines. How well a particular HCP assay recognizes all proteins depends…

Recombinant therapeutic proteins derived from E. coli require robust endotoxin removal, ideally performed using multiple process steps providing orthogonal and robust clearance. Typical recovery processes for E. coli expressed proteins consist of cell harvest by centrifugation, homogenization, collection of inclusion bodies by centrifugation, solubilization, refold, and clarification by depth filtration. Endotoxin clearance across the recovery operations is usually on the order of one log. Here, we demonstrate that endotoxin clearance can be enhanced to five logs by using 16% polyethylene…

Many biotechnological products are obtained by the help of native or recombinant microbial cells in fermentation processes. In order to get intracellular target molecules, like proteins or enzymes, the cells have to be destructed. This is done with cell rupture techniques (e.g. high-pressure homogenization) which generate a suspension containing cell debris and cell contents (proteins, DNA, mRNA, etc.). The first step in the following purification sequence is the removal of the cell debris. One possibility to do this is the…