Systematic development and validation of sanitization protocols for a chromatographic system designed for biotherapeutics purification.

Production of protein therapeutics through the application of genetic engineering and biotechnology techniques requires comprehensive attention to good manufacturing practice and good laboratory practice (GMP/GLP) guidelines for product recovery and purification. Validated clean-in-place procedures are part of the master method and require analysis of microbial bioburden to assess the efficacy of cleaning protocols. This article describes the extensive microbial challenge of a chromatography system, the use of membrane filtration methods for high sensitivity microbial contamination measurement, and the effectiveness of sodium hydroxide and ethanol solutions in achieving multilog reduction of microbial contamination.

Photodiode array spectral identification of modified amino acids in synthetic peptides following high resolution HPLC separation.

The products of peptide synthesis are routinely purified by reversed-phase HPLC with single wavelength detection at 214 nm. Additional information regarding the peptide product and any contaminating sequences can be obtained by high resolution HPLC combined with high sensitivity spectral analysis using photodiode array detection. Full spectral characterization can be used to ascertain the presence of aromatic amino acid residues as well as side chain modifications such as blocking groups for protection of labile side chains. Many of the protecting groups used in solid phase synthesis contain distinctive structural features that can be identified by their spectra. Although the synthesis protocol provides for hydrolysis of these groups, it is possible that the product mixture may include some residual blocked material. Since the presence of blocking groups will affect the properties of the synthesized peptide, detection and complete removal are essential. Spectral analysis using photodiode array detection in conjunction with high resolution HPLC separations can be used to identify protected amino acid residues in synthetic peptides.