Development and scaling of a chromatographic purification technology for single-domain antibodies fused to the Fc fragment of human IgG1

Objectives. The work set out to develop a chromatographic purification technology for drugs based on modified single-domain antibodies specific to the SARS-CoV-2 virus, as well as to select the optimal parameters for the purification process and scale up this technology for production.

Methods. The study was conducted on a culture of Chinese hamster ovary (CHO) cells GAMP2C5  clone 78, CHO B5 clone 4, and CHO B10 clone 4, which were used to produce modified single-domain antibodies GAMP2C5, GAMB5, and GAMB10, respectively. Chromatographic purification was performed using AKTA pure 25 and AKTA Pilot 600s chromatographs. Quality control of the obtained drugs was carried out using high-performance liquid chromatography, capillary gel electrophoresis, dynamic light scattering, enzymelinked immunosorbent assay, and polymerase chain reaction.

Results. Multimodal chromatography using CA⁺⁺Pure-HA (TOSOH, Japan) resin based on type 1 ceramic hydroxyapatite can be effectively used for the removal of aggregated antibody forms. The drugs obtained after chromatography using CA⁺⁺Pure-HA resin based on type 1 ceramic hydroxyapatite have a purity of more than 97%. The developed purification technology was scaled up to purify 200 L of culture fluid after cultivation in an STR 200 bioreactor.

Conclusions. The described technology developed for purifying modified mono-domain antibodies using the CA⁺⁺Pure-HA multimodal resin based on type 1 ceramic hydroxyapatite allows for the effective removal of low-molecular-weight impurities and aggregated forms of the antibody. The antibodies obtained using the developed technology are characterized by a high degree of purity and the absence of various impurities (residual protein of the producer strain, residual protein A, and residual DNA of the producer strain), as well as offering a hydrodynamic molecular radius corresponding to the theoretical value of monomeric forms of antibodies.

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