Increase in cysteine-mediated multimerization under attractive protein-protein interactions.

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein-protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein-protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris.

BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

Protein-protein interactions and reduced excluded volume increase dynamic binding capacity of dual salt systems in hydrophobic interaction chromatography.

Deploying two salts in hydrophobic interaction chromatography can significantly increase dynamic binding capacities. Nevertheless, the mechanistic understanding of this phenomenon is lacking. Here, we investigate whether surface tension or ionic strength govern dynamic binding capacities of the chromatographic resin Toyopearl Butyl-650 M in dual salt systems. Small-angle X-ray scattering was employed to analyze the model proteins and the protein-resin adduct in the respective dual salt systems. The dual salt systems incorporate sodium citrate and a secondary sodium salt (acetate, sulfate, or phosphate). As model proteins, we used lysozyme, GFP, and a monoclonal antibody (adalimumab). Moreover, for the protein-resin adduct, we determined the model parameters of a self-avoiding random walk model fitted into the pair density distribution function of the SAXS data. Ionic strength is more predictive for dynamic binding capacities in HIC dual salt systems than surface tension. However, dynamic binding capacities still differ by up to 30 % between the investigated dual salt systems. The proteins exhibit extensive protein-protein interactions in the studied dual salt HIC buffers. We found a correlation of protein-protein interactions with the well-known Hofmeister series. For systems with elevated protein-protein interactions, adsorption isotherms deviate from Langmuirian behavior. This highlights the importance of lateral protein-protein interactions in protein adsorption, where monomolecular protein layers are usually assumed. SAXS analysis of the protein-resin adduct indicates an inverse correlation of the binding capacity and the excluded volume parameter. This is indicative of the deposition of proteins in the cavities of the stationary phase. We hypothesize that increasing protein-protein interactions allow the formation of attractive clusters and multilayers in the cavities, respectively.