Antibodies are a new class of biological agents that are of increasing interest in immunotherapy applications for tumors. In the construction of antibody formats of different functions, cysteine residues are designed in the heavy and light chain regions of single-chain variable fragments (scFv) to form intrachain disulfide bonds to improve biochemical stability. Structural characterization studies have shown that different size of variants is produced by engineered disulfide bonds on scFv, thereby finding that engineered disulfides are open or due to cysteine or glutathionylation, it does not form an intrachain disulfide bond. In addition, scFv engineered cysteines also form intermolecular disulfide bonds, resulting in the formation of highly stable dimers and aggregates.
Because both monomer variants and dimers exhibit lower biological activity, they are considered to be product-related impurities that must be monitored and controlled. To this end, Creative Biolabs has developed and optimized a robust, accurate, and high-resolution size exclusion chromatography (SEC) method using statistical design experimental methods.
| Services | Techniques |
| Secondary structure | Fourier transform infrared (FTIR) spectroscopy, Circular dichroism |
| Size heterogeneity under native conditions | Size exclusion chromatography (SEC) |
| Size heterogeneity of reducible forms | Reduced capillary sodium dodecylsulfate electrophoresis (rcSDS); reduced, denatured SEC (rdSEC) |
| Size heterogeneity of covalent forms | nrcSDS, dSEC |
| Determination of monomer, dimer, and submicron aggregates | Resolution by SEC with static light scattering detection (SEC-SLS) |
| Size heterogeneity and sedimentation coefficient determination | Sedimentation velocity analytical ultracentrifugation (SV-AUC) |
| Attribute criticality | Purification and characterization of size variants |
Fourier Transform Infrared (FTIR) Spectroscopy (From Wikipedia)
Size heterogeneity under natural solution conditions is typically monitored by non-denaturing SEC, which typically decomposes submicron aggregates (i.e., HMW species) from monomers in the main peak. Low molecular weight (LMW) materials are generally poorly separated from the excipient peaks and/or co-eluted and can be better monitored by capillary electrophoresis with or without reduced sodium dodecyl sulfate (rcSDS or nrcSDS). Examples of common protein size modifications commonly detected by the SEC are:
Monomer and submicron aggregates can be purified by fraction collection from peaks of the SEC method, and subsequent analyses such as cSDS and rcSDS can help elucidate the covalent nature of submicron aggregates.
Feel free to contact us for project quotations and more detailed information.
For Research Use Only | Not For Clinical Use
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