Wash with at least 2 CV distilled water.ĥ. Wash with at least 3 CV 1 M NaOH with at least 15 min contact time.Ĥ. Wash with at least 2 column volumes (CV) of 2 M NaCl.Ģ. Stable to commonly used aqueous buffers, 1M acetic acid, 1.0 M NaOH, 8 M Urea, 6 M guanidine hydrochloride, 70% ethanol, 30% isopropanol.ġ. This was also obtained on a Capto Q ImpRes resin. In conventional AEX resins, the dsDNA fragments provide a stronger interaction compared with ssDNA. HiTrap Capto MMC can be used to bind proteins at the conductivity of the feed material and to solve specific purification challenges. In this study, we have compared a strong AEX resin, Capto Q ImpRes, with an MMC resin ( Capto Adhere) from previous work 7. Selectivity allows binding in the presence of salt. High dynamic binding capacity at high conductivity. To reduce tedious sample preparation, the top filter in HiTrap Capto MMC has been optimized for loading sonicated unclarified cell lysate directly on the column. This means that the resin may be used for direct load of clarified feedstocks, without prior dilution to reduce the conductivity of the starting material. The multimodal functionality gives a different selectivity compared with traditional ion exchangers including binding of proteins regardless of ionic strength of the loading material. The multimodal functionality gives a different selectivity compared with traditional ion exchangers and it binds proteins at high or low ionic strength. All rights reserved.HiTrap Capto MMC is prepacked with Capto MMC, a multimodal BioProcess ion exchange resin. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.Īntibody Fragment removal Mixed mode resin.Ĭopyright © 2017 The Author(s). Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility.
CAPTO MMC RESIN PATCH
Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. Further decreases in LHF separation were seen upon incubation with urea and arginine. The addition of ethylene glycol decreased LHF removal by half.
CAPTO MMC RESIN SERIES
The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. The mechanism of fragment removal was investigated in two series of experiments. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing.
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