Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain.
Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The CH3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the CH3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the CH3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetry (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules.
High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan; Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: sakurai@waka.kindai.ac.jp.
Kazumasa
K
Sakurai
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Ryosuke
R
Nakahata
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Young-Ho
YH
Lee
Department of Biochemistry, Eötvös Loránd University, Pázmány P. sétány 1/C, Budapest, H-1117 Hungary; MTA-ELTE NAP B Neuroimmunology Research Group, Eötvös Loránd University, Pázmány P. sétány 1/C, Budapest, H-1117 Hungary.
József
J
Kardos
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Takahisa
T
Ikegami
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Yuji
Y
Goto
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D066329:Protein Aggregates; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D066329:Protein Aggregates; D011994:Recombinant Proteins
Aggregation; Calorimetry; Disulfide; Immunoglobulin fold; NMR