In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme\'s specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme\'s isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 μg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.

The drug product development process for proteins went through its infancy in the early eighties of last century and is in its maturity today. This has been driven largely by the rapid growth of the biotechnology industry, which led to the development and issuance of many regulatory guidelines/directories, especially those through the International Conference of Harmonization (ICH). These guidelines have certainly guided different aspects of a drug product development process. On the other hand, they were issued separately on different topics and in different time periods. An integration of all relevant guidelines into the corresponding areas in drug product development would greatly facilitate the development process. The purpose of this short review is to integrate the relevant (mainly ICH) regulatory guidelines into protein drug product development and to discuss remaining issues, which may lead to further revision of existing guidelines or development of new ones. Drug product development scientists need to collect adequate and relevant development data for a successful product registration. The key is the ability to justify the final drug product in terms of choice of the drug product formulation, container closure system, and manufacturing process.
The drug product development process for proteins has matured today, largely due to the rapid growth of the biotechnology industry. In this process, many regulatory guidelines/directories were developed and issued, especially through the International Conference of Harmonization (ICH). However, they were issued separately on different topics and in different time periods. An integration of all relevant guidelines into the corresponding areas in drug product development would greatly facilitate the development process. The purpose of this short review is to integrate the relevant (mainly ICH) regulatory guidelines into protein drug product development and to discuss remaining issues, which may lead to further revision of existing guidelines or development of new ones. Drug product development scientists need to collect adequate and relevant development data for a successful product registration. The key is the ability to justify the final drug product in terms of choice of the product formulation, container closure system, and manufacturing process.