No abstract

This work concerns the chromatographic separation of protein charge variants using pH gradients generated by step changes in buffer composition with weak base anion exchange columns. A local equilibrium model is first developed to describe pH transitions occurring in the column using buffers comprising neutral, zwitterionic or positively charged species. Model predictions, based solely on the resins' titration curves and obtained with the method of characteristics, show, in excellent agreement with experiments, that induced pH gradients of varying durations and shapes can be obtained with a broad range of buffer systems including Tris, Bis-Tris propane, histidine, and their mixtures and ethanolamine. The separation of protein charge variants is then demonstrated for bovine apo-transferrin and for a monoclonal antibody. The resolution of the charge variants present in these proteins, demonstrated via isoelectric focusing analyses, is obtained for conditions amenable to scale-up for preparative purposes; that is larger particle sizes (90 lm), higher flow rates (100-600 cm/h), and higher protein loads (2-5 mg/mL). Because the approach requires only step changes in buffer composition and commonly available, unretained buffers species, practical implementation is straightforward. The focusing effect of the induced pH gradient results in relatively sharp peaks and substantial resolution even for these conditions.

The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.

No abstract

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A-I(Milano) (apo A-I(M)) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale-up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co-immunoprecipitation and Western blotting techniques. Two-dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory-scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign.