Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability
Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability
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DOI：
10.1074/jbc.m112.369744
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发表时间：
2012-07-13
影响因子：
4.8
通讯作者：
Humphreys, David P.
中科院分区：
文献类型：
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作者：
Peters, Shirley J.;Smales, C. Mark;Humphreys, David P.
随着抗体受到监管当局越来越多的审查，抗体结构的完整性、稳定性和生物物理特性变得越来越重要。我们通过将重链恒定域1(C(H)1)N端的半胱氨酸(Kabat位置127)突变为Ser，并在C(H)1的C末端的不同位置(227-230位)引入半胱氨酸，从而改变了IgG4分子的二硫键排列，从而形成了与IgG1分子中的二硫键排列相似的二硫键。在中国仓鼠卵巢细胞瞬时表达后，用免疫印迹分析上清液中的抗体种类以确定产物的同源性，并用热荧光法分析纯化产物的热稳定性。我们表明，当轻链存在于上铰链上的几个位置(位置227-230)时，轻链可以与半胱氨酸形成LC-C(H)1二硫键之间的二硫键，因此这种工程化的二硫键可以提高FAB结构域的热稳定性，在3-6.8℃之间。IgG4二硫键突变体在FAB热稳定性方面表现出最大的提高，也是在二硫键排列和抗体种类方面最均匀的。重要的是，突变不会影响所产生的分子对抗原的亲和力。结合前面描述的S241P突变，我们提出了一种具有更高的Fab热稳定性和更低的产物异质性的IgG4分子，这可能为生产IgG4分子提供优势。
The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 degrees C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.