Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies.
Temperature-induced unfolding of three humanized IgG1 monoclonal antibodies and their Fab and Fc fragments was monitored by differential scanning calorimetry at neutral pH. With some exceptions, the thermogram of the intact antibody presents two peaks and the transition with the larger experimental enthalpy contains the contribution from the Fab fragments. Although the measured enthalpy was similar for all three Fab fragments studied, the apparent melting temperatures were found to vary significantly, even for Fab fragments originating from the same human germline. Therefore, we propose to use the measured enthalpy of unfolding as the key parameter to recognize the unfolding events in the melting profile of an intact IgG1 antibody. If the variable domain sequences, resulting from complementarity determining regions (CDRs) grafting and humanization, destabilize the Fab fragment with respect to the CH3 domain, the first transition represents the unfolding of the Fab fragment and the CH2 domain, while the second transition represents CH3 domain unfolding. Otherwise, the first transition represents CH2 domain unfolding, and the second transition represents the unfolding of the Fab fragment and the CH3 domain. In some cases, the DSC profile may present three transitions, with the Fab unfolding occurring at distinct temperatures compared to the melting of the CH2 and CH3 domains. If the DSC profile of a humanized IgG1 monoclonal antibody cannot be described by the model above, the result may be an indication of significant structural heterogeneity and/or of disruption of the Fab cooperative unfolding. Low stability or heterogeneity of the Fab fragment may prove problematic for long-term storage or consistency of production. Therefore, understanding the features of a DSC profile is important for clone selection and process maturation in the early stages of development of therapeutic monoclonal antibodies.
Ionescu
Roxana M
RM
Biologics and Vaccines PR&D, Merck Research Laboratories, Merck and Co., Inc., West Point, Pennsylvania, USA. roxana_ionescu@merck.com
Vlasak
Josef
J
Price
Colleen
C
Kirchmeier
Marc
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D022801:Complementarity Determining Regions / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G