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The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

Host cell proteins (HCPs) are process-related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Some have been shown to be difficult to remove by purification. Why should the biopharmaceutical industry worry about these high-risk HCPs? What approach could be taken to understand the origin of its copurification and address these *Marisa Jones and Nisha Palackal should be considered joint first authors About Biophorum Development Group (BPDG): Since its inception in 2004, BioPhorum has become a trusted environment in which senior leaders of the biopharmaceutical industry come together to share and discuss openly the emerging trends and challenges facing their industry.BioPhorum currently comprises more than 3800 active participants in seven "phorums" covering cell and gene therapy, drug substance, development, fill-finish, a technology roadmap, information technology, and supply partners. The Host Cell Protein (HCP) Workstream is part of the Development Group (BPDG). This article is a composite view of opinions shared by the whole of the BPDG-HCP Workstream and should not be attributed to the individual positions of the participating companies.

We have determined the macromolecular composition of inclusion bodies formed by overexpressing P-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition.Specifically, contaminating polypeptides ranged from less than 5 % to over 50% of the total protein content. Phospholipids composed 0.5-13 % of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature P-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown a t basic pH. Our results show that, at least under some growth conditions, protein aggregation i n vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

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