Acid-induced aggregation propensity of nivolumab is dependent on the Fc.
Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.
a School of Bioscience and Bioengineering , South China University of Technology , Guangzhou , China.|b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Boning
B
Liu
c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.|d Shanghai Zhangjiang Biotechnology Co. , Shanghai , China.
Huaizu
H
Guo
c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.|d Shanghai Zhangjiang Biotechnology Co. , Shanghai , China.
Jin
J
Xu
a School of Bioscience and Bioengineering , South China University of Technology , Guangzhou , China.|b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Ting
T
Qin
a School of Bioscience and Bioengineering , South China University of Technology , Guangzhou , China.|b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Lu
L
Xu
a School of Bioscience and Bioengineering , South China University of Technology , Guangzhou , China.|b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Junjie
J
Zhang
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Qingcheng
Q
Guo
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Dapeng
D
Zhang
c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.|d Shanghai Zhangjiang Biotechnology Co. , Shanghai , China.
Weizhu
W
Qian
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Bohua
B
Li
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Jianxin
J
Dai
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.
Sheng
S
Hou
a School of Bioscience and Bioengineering , South China University of Technology , Guangzhou , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.|e School of Pharmacy , Liaocheng University , Liaocheng , China.
Yajun
Y
Guo
b International Joint Cancer Institute , Second Military Medical University , Shanghai , China.|c State Key Laboratory of Antibody Medicine and Targeted Therapy , Shanghai Key Laboratory of Cell Engineering ; Shanghai , China.|e School of Pharmacy , Liaocheng University , Liaocheng , China.
Hao
H
Wang
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry; D000920:Antibody-Dependent Cell Cytotoxicity; D000970:Antineoplastic Agents / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007132:Immunoglobulin Isotypes / Q000737:chemistry*; D008353:Mannitol / Q000737:chemistry; D000077594:Nivolumab; D000077544:Panitumumab; D057216:Propensity Score; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D000077594:Nivolumab; D008353:Mannitol; C000594389:atezolizumab; D000077544:Panitumumab; C502936:tocilizumab
Acid-induced aggregates; Fc; IgG4 subclass; Protein A chromatography; atezolizumab; nivolumab; panitumumab; purification process; tocilizumab; viral inactivation