ABSTRACT
BACKGROUND: Recent advancements in cell engineering and bioreactor engineering have enabled high monoclonal antibody (mAb) concentrations in harvested solutions for the downstream process (DSP).
Methods: As many unit operations such as capture chromatography, polish chromatography, membrane filtration, virus inactivation, virus filtration, and concentration by ultrafiltration are involved in DSP, it is crucial to monitor the process carefully in order to perform reliable and stable DSP operations. One of the most important signals (process parameter) to be monitored is the protein concentration CP. Although various methods are available, most of them are not suited for measuring high CP. In this paper, we have developed a method for measuring very high CP by optical rotation (OR).
Result: Linear correlations were confirmed between OR and Cp in the range CP = 0 to 80 g/L for mAbs with high repeatability and small variation coefficients. This method was applied to the monitoring of CP in the opaque (colored) solution during the cell culture. The CP by OR was in good agreement with those by the standard Protein A HPLC method.
Conclusion: Monitoring of high CP by OR is expected to be an efficient process analytical tool (PAT) for DSP.
Subject(s)
Antibodies, Monoclonal , Bioreactors , Antibodies, Monoclonal/chemistry , Chromatography, High Pressure Liquid , Optical RotationABSTRACT
Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M. (1995) FEBS Lett. 372, 210-214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography. Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI. Screening for hPDIR-binding proteins using a T7 phage display system revealed that alpha1-antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that alpha1-antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of alpha1-antitrypsin with hPDIR was much higher than with hPDI or hP5. Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of alpha1-antitrypsin was the most definite of the three (CSMC, CGHC, CPHC). Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding. In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding. Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent. These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.
Subject(s)
Proteins/chemistry , Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , DNA, Complementary/genetics , Humans , In Vitro Techniques , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Binding , Protein Disulfide-Isomerases/metabolism , Protein Folding , Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , alpha 1-Antitrypsin/metabolismABSTRACT
Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.
