Method for repairing protein drug fragments in antibody production process
A repair method and technology of production process, applied in the fields of bioinformatics, instruments, electrical digital data processing, etc., can solve the problems of few remedies and poor effect of reduction products
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Embodiment 1
[0077] Example 1 Based on the kinetic model above, the effect of protein A on the reoxidation of disulfide bonds was studied
[0078] Protein A is a 42kDa surface protein that is used as a resin after harvest to capture IgG. Due to its high binding affinity to the Fc region of the heavy chain, it is highly selective for IgG type antibodies. Such as figure 2 As shown, the fragment molecules were slowly re-oxidized in pH 8 buffer. The presence of protein A resin accelerates the reoxidation process by accelerating all fundamental reactions (see figure 2 ). The mechanism is that the protein A resin traps and concentrates the debris on the resin surface, thereby reducing the activation energy of the reoxidation reaction.
Embodiment 2
[0079] Example 2 Based on the above-mentioned kinetic model, the influence of conductivity on the reoxidation of disulfide bonds was studied
[0080] Conductivity is an important property of buffer systems. It needs to be well controlled during production. In this method, sodium chloride is used to adjust the conductivity of the buffer. The kinetics at different conductivities were then measured to assess the impact. Conductivity was found in this example to have a negative impact on reoxidation kinetics (see image 3 A and 3B). That is, higher reoxidation rates were observed at lower conductivities. Conversely, at higher conductivity, the rate of reoxidation is slower. This inverse relationship between reoxidation rate and solution conductivity may be due to the negative effect of salt concentration on molecular interactions [1，2] . Table 1 shows that protein A resin accelerates the reaction at different conductivities than the respective resin-free conditions. To qua...
Embodiment 3
[0084] Example 3 Based on the kinetic model above, the effects of different redox agents and pH on the reoxidation of disulfide bonds were studied
[0085] Cysteine, cystine and glutathione can be used as mild redox reagents for the repair of protein drug fragmentation. Its redox ability can be adjusted by controlling the pH of the buffer system. Its specific role can be designed through DoE, and the application software JMP 13 can be used for statistical analysis.
[0086] As shown in Table 2, the final purity of this embodiment changes under different conditions. The purity at pH 7 was lower than that at pH 8 and pH 10, suggesting that for the reoxidation process, alkaline conditions are preferred, and the mechanism is due to the chemical potential change at different pHs. While this material had an initial purity of 64%, when examined on the Protein A column alone (without any of the above chemicals), the purity increased to around 85% at pH 8 and 10. This again shows th...
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