![]() In summary, this study offers insight into the mechanism of mAb aggregation in bind-elute CEX operations, and the in-depth understanding facilitates the development of robust CEX conditions for mAb purification.Īggregate formation Cation-exchange chromatography Hydrophobicity Monoclonal antibody Structural stability.Ĭopyright © 2020 Elsevier B.V. Results suggest that the mAb-accessible hydrophobic regions of the CEX resins affect the structural stability of the bound mAbs to various degrees, leading to differences in aggregate formation upon mAb elution. HiScreen Capto SP ImpRes column is packed with strong cation exchange modern resin for fast high resolution protein purification in process development. Capto Q O CHN(3) 3 OH + Strong anion exchange group. Resin type Active group Description Capto S O SO-3 Strong cation exchange group. Table 1.The active groups of Capto S, Capto Q and Capto DEAE. Finally, the hydrophobicity of the CEX resins (Capto SP ImpRes < Fractogel EMD SE Hicap < POROS XS) was measured using a fluorescence-based method to quantitatively characterize this resin property. group, Capto Q uses a quartenary amine group and Capto DEAE uses a diethylaminoethyl group, as shown in the table below. The interplay among these protein- and resin-related factors, together with solution conditions, ultimately dictates the aggregate formation observed. In particular, resin hydrophobicity was shown to have a critical impact. The Tm differences (∆Tm DSC (Unbound minus Bound)) between the two states correlated with the severity of mAb aggregation in CEX operations, indicating the importance of both intrinsic mAb stability and resin properties. Using differential scanning calorimetry (DSC), the measured melting temperature of the bound mAbs (Tm DSC (Bound)) was 4.5 - 6.5☌ lower than that for the unbound mAbs (Tm DSC (Unbound)) in the same solutions. Then, mAb structural stability was further investigated in the bound state on CEX surfaces. Using differential scanning fluorimetry (DSF), the measured melting temperature (Tm DSF (Unbound)) decreased from 60.7 to 52.4☌ for mAb1 and 51.5 to 45.2☌ for mAb2 when lowering pH from 6.0 to 4.5. First, mAb structural stability was studied in solutions under CEX load conditions. ![]() Running buffer for MAb B: 20 mM sodium acetate, pH 5.0. DBC MAb at 5 break through (mg/mL of resin) Figure 2B: 5 DBC for MAb B at two residence times on Praesto SP65, Praesto SP45, Capto SP ImpRes and Capto S Impact. ![]() Running buffer for MAb A: 20 mM sodium acetate, pH 5.5. To gain mechanistic understanding of this phenomenon, aggregate formation in bind-elute CEX for two therapeutic mAbs (IgG1 and IgG4) was examined on three CEX resins (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS). Praesto SP65, Praesto SP45, Capto SP ImpRes and Capto S Impact. High molecular weight (HMW) aggregate formation of therapeutic monoclonal antibodies (mAbs) during cation-exchange chromatography (CEX) has been frequently observed, and can be a challenge for downstream purification. ![]()
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