Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assay.

Determination of excipient content in drug formulation is an important aspect of pharmaceutical formulation development and for analytical testing of the formulation. In this study, the influence of polysorbate subspecies, in particular mono- and poly-esters, for determining polysorbate (PS) content were investigated by comparing three of the most widely used PS quantitation approaches, the Fluorescence Micelle Assay (FMA) and Mixed-Mode High Performance Liquid Chromatography coupled with Charged Aerosol Detection (MM-CAD) or Evaporative Light Scattering Detection (MM-ELSD). FMA and MM-CAD were employed to investigate the quantitation behavior of PS20 and PS80 subspecies and corresponding degradation products in placebo formulations using forced degradation conditions at 40°C for up to 12 weeks. While both methods allowed accurate and comparable quantification of neat PS at the beginning of stress studies, pronounced differences in content determination between the methods were observed at later time points, which were attributable to substantial differences in the contribution of individual mono- and poly-esters to the overall quantitation results. It was particularly surprising to find that the main component of PS20, polyoxyethylene sorbitan monolaurate, did not show a signal at the studied concentration using FMA. Moreover, the degradation of polysorbate poly-esters, was reflected much stronger in FMA than MM-CAD results. Additional experiments employing chemical oxidation and base hydrolysis to degrade PS20, quantified by FMA and MM-ELSD, also show preferential reduction in certain subspecies depending on the degradation pathway involved. For PS20 degraded by chemical oxidation, quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with base hydrolysis.

The immunogenicity of antibody aggregates in a novel transgenic mouse model.

PURPOSE: Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations. METHODS: Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light. RESULTS: The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic. CONCLUSION: This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.

Structure and function of purified monoclonal antibody dimers induced by different stress conditions.

PURPOSE: To investigate structure and function of different monoclonal antibody (MAb) dimers. METHODS: MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods. RESULTS: Highly purified dimer forms were obtained which enabled detailed characterization. Dimers induced by process stress were associated by a single non-covalent interaction site between two Fab domains in a characteristic "bone-like" structure observed in Transmission Electron Microscopy (TEM). These dimers showed reduced potency and antigen binding affinity. Low pH stress generated more stable but also non-covalently associated dimers without chemical alterations in a typical "closed" conformation according to TEM. These dimer species were more compact and more hydrophobic as dimers induced by process stress. They showed bioactivity and antigen binding affinity similar to the native monomer. Light-induced dimers, exhibiting various different conformations, were the most stable dimers with various chemical modifications leading to a broad range in size, charge and hydrophobicity. These dimers fully lost bioactivity and antigen binding affinity. CONCLUSION: The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: characterization of biocatalytically active bacterial strains and of cytochrome p450 monooxygenase enzymes and their genes.

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: heterologous expression of the ema1 cytochrome P450 monooxygenase.

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.