Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6-derived recombinant antibody.

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow-through mode for contaminant removal. As flow-through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind-and-elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow-through applications. Given these considerations, a new Sartobind Phenyl membrane adsorber was developed for large-scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography-virtually no diffusion limitation and shorter processing time-with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg-MAb/cm(3)-membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five- to ten-fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6(R) cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale-up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography.

A chemical approach to the pharmaceutical optimization of an anti-HIV protein.

Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site-specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL-5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-HIV therapeutics which are still desperately needed.