Removing a light-chain missing byproduct by MMC ImpRes mixed-mode chromatography under weak partitioning mode in purifying a WuXiBody-based bispecific antibody
Introduction
WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics [1]. This platform enables any monoclonal antibody sequence pair to be assembled into a bispecific construct. Its key feature is the replacement of one parental antibody's CH1/CL region with the T cell receptor (TCR) constant domain, which promotes cognate heavy chain (HC)-light chain (LC) pairing. WuXiBody's sequence replacement has a similar effect as domain crossover in the CrossMab approach [2]. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format [1,3].
While purifying a symmetric WuXiBody-based bsAb, we identified a LC-missing species as a major byproduct. In particular, the lost LC is exclusively the modified one that contains the TCR constant domain (Fig. 1). LC-missing byproduct was also identified as an impurity during the production of CrossMab [2]. It is likely that in both cases (i.e., WuXiBody and CrossMab) the sequence engineered Fab arm is less stable and therefore a small portion of the bsAbs may lose their LC in that arm. Removing this LC-missing species can be difficult, as such a byproduct usually shares high similarity in physicochemical properties with the intact bsAb. Fortunately, WuXiBody's unique design facilitates removal of this type of byproduct. In WuXiBody, the introduced TCR constant domain has a relatively low isoelectric point (pI). Consequently, the byproduct, which loses the TCR-containing LC, has a pI higher than that of the product (Fig. 1), and such pI difference facilitates separating the LC-missing byproduct from the intact bsAb by ion exchange (IEX) or mixed-mode (IEX and hydrophobic interaction) chromatography.
For the symmetric WuXiBody-based bsAb mentioned above, we previously demonstrated that the LC-missing byproduct can be readily separated from the intact bsAb using mixed-mode resin Capto MMC ImpRes, whose ligand possesses cation exchange (CEX) and hydrophobic moieties [4,5]. As the LC-missing byproduct has a pI slightly higher than that of the intact bsAb (5.73 vs. 5.52), it binds to the MMC ImpRes column stronger than the product [6]. While relatively complete separation between these two species was achieved under bind-elute mode with either pH or salt gradient elution, the column loading density was relatively low (i.e., 20 mg of protein per ml of resin). Such a low loading density is unfavorable for manufacturing at large scale. Although a lower loading pH (<6.0) can slightly increase the dynamic binding capacity (DBC), it triggers precipitation of the product in this case. In order to solve this dilemma, in this study we evaluated the feasibility of switching from bind-elute mode to weak partitioning mode using the same resin. The data suggest that under weak partitioning mode high throughput, good yield, and effective byproduct removal are simultaneously achieved. This finding indicates that in certain case weak partitioning mode could be a better alternative than bind-elute mode for removing impurity that binds slightly tighter than the product.
Section snippets
Materials
Disodium hydrogen phosphate dihydrate, sodium acetate trihydrate, sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, tris(hydroxymethyl)aminomethane and ethanol were purchased from Merck (Darmstadt, Germany). Acetic acid and arginine hydrochloride were purchased from J.T.Baker (Phillipsburg, NJ, USA). Iodoacetamide and SDS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Precast SurePAGE 4–12% gradient Bis-Tris gels, liquid container and eStain LG protein destainer
Protein A chromatography
MabSelect SuRe LX was packed in a 2.6 cm diameter column with 20.0 cm bed height. The column volume (CV) is approximately 106 ml. The column was loaded at 28 mg of protein per ml of resin. After loading, the column was washed consecutively with 50 mM Tris-HAc, 150 mM NaCl, pH 7.4; 50 mM NaAc-HAc, 1 M NaCl, pH 5.5 and 50 mM NaAc-HAc, pH 5.5; each for 3 CV. The column was eluted with 50 mM NaAc-HAc, pH 3.7.
Capto MMC ImpRes chromatography
Capto MMC ImpRes chromatography was performed in bind-elute or weak partitioning mode using
Major impurity post protein A chromatography
Product was captured from clarified culture harvest by Protein A chromatography following standard protocol as described in the Method section (chromatogram not shown). The column eluate was analysed by non-reduced Caliper, and the profile suggests the existence of a major impurity whose molecular weight is slightly lower than that of the target product (Fig. 2). Further analysis by mass spectrometry indicates that this low-molecular-weight species is the product losing one TCR-containing LC
Conclusion
In a previous study, we showed that, when purifying a symmetric WuXiBody-based bsAb, Capto MMC ImpRes mixed-mode chromatography under bind-elute mode can effectively remove a LC-missing byproduct [6]. However, it was learned that DBC of this resin was relatively low under the selected condition. Increasing DBC by lowering loading pH is impractical in this case as the target bsAb becomes less stable at pH below 6.0. In the current study, we demonstrated that switching from bind-elute mode to
Author statement
Ting Zhang: Data curation, Formal analysis, Investigation, Writing - review & editing. Yan Wan: Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Jiwei Duan: Data curation, Formal analysis, Writing - review & editing. Yifeng Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing.
Acknowledgements
We would like to thank Weichang Zhou, Peter (Keqiang) Shen, Gang Huang and Ying Wang for lending their support to this work. We thank Technology and Process Development (TPD) Department's cell culture team for material generation. We also thank Guoqiang Xu for assistance with non-reduced Caliper.
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2022, Protein Expression and PurificationCitation Excerpt :In general, mixed-mode resins offer selectivity different from that of single-mode resins. Recently mixed-mode resins have found increased use as a polishing step for bispecific antibody purification [32–34]. In comparison to single-mode resins, mixed-mode resins often display high and robust viral clearance over a broader range of pH and conductivity.
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These two authors contributed equally to this work.