Acid-Induced Aggregation of Human Monoclonal IgG1 and IgG2: Molecular Mechanism and the Effect of Solution Composition

Abstract

The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.

Human IgG1, IgG3, and IgG3 Hinge-Truncated Mutants Show Different Protection Capabilities against Meningococci Depending on the Target Antigen and Epitope Specificity

“…In brief, the bacterial cells were grown in Mu-eller-Hinton broth to mid-log phase, harvested, and resuspended in buffer as described elsewhere (38). Immediately before the assay was performed, the MAbs were centrifuged for 2 h at 100,000 rpm to remove possible aggregates, since the aggregation of antibodies that occurs during development and storage may lead to the loss of biological activity (39). The 40-l bactericidal reaction mixture contained 1 to 100 g of MAb/ml, ca.…”

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

“…In addition to effector functions and stability and biophysical characteristics, analytical and regulatory aspects are important selection criteria for such monoclonal antibodies (mAbs) destined for clinical use. Further, it is well known that during manufacturing, purification, formulation, and storage, the isotypes behave differently in terms of their sensitivity to pH or temperature, aggregation propensity, and susceptibility to degradation (11)(12)(13). With regard to monitoring or predicting stability, thermal stability is increasingly accepted as a convenient surrogate measure of global stability.…”

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

“…To accelerate bevacizumab aggregation, the incubation temperature was increased. It has previously been shown that when bevacizumab at concentrations below 2.5 mg/mL (pH 6.0-6.2) is incubated at 56 C, 65 C and 70 C, almost all of the protein aggregates within 3 days, 3 h and 10 min, respectively. 21,44 To minimize unfolding of bevacizumab at such a high temperature (temperature close to Tm), a lower temperature was used for these accelerated studies.…”