There are various methods available for assessing the stability of protein formulations, incluidng antibody formulations, based on the physical and chemical structures of the proteins as well as on their biological activities. Methods include charge-transfer adsorption, thermal analysis, fluorescence spectroscopy, circular dichroism (CD), NMR, and HPSEX, tangential flow filtration (TFF), static light scattering (SLS), fourier transform infrared spectroscopy (FTIR), urea induced protein unfolindg techniques, intrinsic tryptophan fluorescence, differential scanning calorimety and I-anilino-8-naphthalenesulfonic acid (ANS) prtoein binding techniques. (Carpenter, WO 2008/039761).
Forced degradation assays of mAbs are frequently performed to support formulation development of mAbs. The commonly used forced degradation conditions include high temperature, freez-thaw, agitation, high pH, low pH, light exposure, oxidation and glycation. SEC is the method of choice to detect aggregation and to a lesser degree, fragmentaiton. Charge-based methods such as CEX or isoelectirc focusing electrophoresis are very sensitive to the detection of overall changes to the molecule, mianly due to deamidation but from other modificaiton as well. Liquid chromatography-mass spectrometry (LC-MS) analysis of the intact or reduced MW is also very useful for detection of modificaitons such as oxidation and glycation. (Liu, “Forced degradation of recombinant monoclonal antibodies: a practical guide” MABS 2017, 9(8) 1217-1230).
Monitoring Protein Aggregates
reducing capillary gel electrophoresis (rCGE) and high performance size exclusion chromatography (HPSEC) are the most common and simplest methods to assess the formation of protein aggregates, protein degradation and protein fragmentation. For example, the percent area of the peaks represent the non-degraded antibody or non-degraded antibody fragments. (Carpenter, WO 2008/039761; Bishop (US 2010/0209434))
Charge Variants
Ion Exchange High Performance Liquid Chromatography (IEX-HPLC): charge variants can be separated by IEX-HPLC. The relative amount of a specific protein impurity, expressed as area %, can be calculated by dividing the peak area corresponding to the impurity by the total integrated area. (Brige (US 2014/0178383)
–Cation-exchagne high performance liquid chromatography (cIEX-HPLC): is a technique that relies on charge-charge interactions between a protein and the charges immobilized on the resin. It takes advantgage of the positvely charged ions of a protein that bind to the engatively charged resin. A common structural modificaiton is deamidation of asparagine (Asn) residues and cEX-HPLC permits the separation of deamidated and deamidation intermediates. (Hays US 2008/0113914)