Light chain subunit of a poorly soluble human IgG2λ crystallizes in physiological pH environment both in cellulo and...

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody's solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a "physiological test tube" that not only reports secretory cargo's high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.