The beneficial impact of kosmotropic salts on the resolution and selectivity of Protein A chromatography.

During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.

A fast and sensitive high-throughput assay to assess polysorbate-degrading hydrolytic activity in biopharmaceuticals.

Hydrolysis of polysorbate in biopharmaceutical products has been ascribed to the enzymatic activity from trace levels of residual host cell proteins. In recent years, significant efforts to identify the causative enzymes typically used elaborate, material-intensive and time-consuming approaches. Therefore, the lack of fast and sensitive assays to monitor their activity remains a major bottleneck for supporting process optimization and troubleshooting activities where time and sample throughput are crucial constraints.

Implementation of in vitro glycoengineering of monoclonal antibodies into downstream processing of industrial production.

In vitro glycoengineering using exoenzymes for specific modification is recognized as appropriate method to tailor sugar moieties of glycan structures during the recombinant production of monoclonal antibodies (mAbs). This report describes enhanced in vitro glycoengineering approaches using β1,4-galactosyltransferase and α2,6-sialyltransferase to improve the efficiency of galactosylation and sialylation with the aim to implement in vitro glycoengineering into common mAb purification processes. Feasibility studies tested the potential of different in vitro glycoengineering protocols (two-step vs.

Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation.

Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis.

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab.

Role of urea on recombinant Apo A-I stability and its utilization in anion exchange chromatography.

Apolipoprotein A-I (Apo A-I) is an important lipid-binding protein involved in the transport and metabolism of cholesterol. High protein purity, in particular with respect to endotoxins is required for therapeutic applications. The use of urea during the purification process of recombinant Apo A-I produced in Escherichia coli has been suggested so as to provide high endotoxin clearance.

Synthesis and characterization of a chimeric peptide derived from fasciculin that inhibits acetylcholinesterase.

Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally.