Analytical Sciences

Factors to Govern Soluble and Insoluble Aggregate-formation in Monoclonal Antibodies

Jun FUKUDA,*,** Takafumi IWURA,** Shigehiro YANAGIHARA,** and Kenji KANO*

*Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan
**Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara, Takasaki, Gunma 370-0013, Japan

The aggregation formation of monoclonal antibodies as biopharmaceuticals induced by heat stress was evaluated by size-exclusion chromatography, and the formation rate was correlated with several physicochemical parameters of the antibodies to clarify the factors to govern the aggregate formation. The parameters we studied were: the melting temperature (T_m) and the standard enthalpy of the melting point (Δ_mH°) evaluated by differential scanning calorimetry under given and common conditions; the wavelength (λ_max) and the intensity (F_int) of the maximum fluorescence peak of 1-anilinonaphthalene-8-sulfonate as a probe dye; the z-average diameter (D) evaluated by dynamic light scattering; and the isoelectric point (pI) and the hydrophobic index (H_pho) of the complementarity determining region calculated from the amino acid sequence. Multivariate statistical analysis with these explanatory variables based on Akaike’s information criterion indicates that the soluble aggregate formation is negatively correlated with T_m and pI, while the insoluble aggregate formation is positively correlated with F_int and pI. Based on these results, the mechanisms of the aggregate formation and methods to prevent the formation are discussed.

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