Data gathered after the incubation period consisted of host cell protein and thioredoxin concentrations remaining in the supernatant, which was considered flowthrough for the remainder of this study. Thioredoxin containing lysate was dialyzed into the binding conditions, pH from 5.0 to 9.0 and conductivity from 2.0 to 10.0 mS, applied to each resin and incubated with shaking for 0.5 h. coli was cultivated, harvested, and lysed according to Ravi et al. Five chromatography resins were screened using a 96-well filter plate to experiment on a wide range of pH and conductivity conditions in a shorter amount of time while saving on materials. This data was used by “Capture chromatography with mixed-mode resins: A case study with recombinant human thioredoxin from Escherichia coli” to determine the optimal resin to use as a capture step to initiate downstream processing of thioredoxin. This paper provides the data collected from screening chromatographic resins for their ability to bind and purify recombinant human thioredoxin from Escherichia coli lysate. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Further investigation revealed that CH2 clippings at 元06 and 元09 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. ![]() Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or 元09 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase – liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. ![]() 12 used three of these approaches (in-gel digestion, RPLC fractionation, and gel-free fractionation) to investigate an unknown 10-kDa fragment and a concomitant shoulder of the monomer peak in nonreduced CE-SDS (nrCE-SDS) of a heat-stressed mAb.įragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis–sodium dodecyl sulfate (CE-SDS). Owing to difficulties in direct fraction collection or online coupling to a mass spectrometer, studies on CE-SDS peak identification largely rely on: 1) prior knowledge of possible species under certain conditions 2) sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) separation, gel band excision, and in-gel digestion peptide mapping 3) gel-free fractionation, intact mass, and peptide mapping 12 4) SEC-based fractionation with offline intact mass, peptide mapping, and CE-SDS 10,13,14 and 5) reversedphase liquid chromatography (RPLC) based fractionation, intact mass, top-down tandem mass spectrometry (MS/MS), or offline peptide mapping and CE-SDS. In contrast, capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) methods are better suited for fragment quantitation at all stages of pharmaceutical development for lot release, stability, in-process testing, characterization, and investigations.
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