Characterization and Quantitation of Antibody Aggregates by High Mass MALDI Mass Spectrometry.

RP-49 Size Exclusion Chromatography (SEC) fractions of therapeutic antibody aggregates were collected and submitted to intact analysis by High-Mass MALDI (Matrix Assisted Laser-Desorption / Ionization) mass spectrometry. SEC fractions were stabilized using MALDI crosslinking (K200 Stabilization Kits, CovalX, Zurich, Switzerland) followed by High-Mass MALDI mass spectrometry (HM2, CovalX, Zurich, Switzerland) for intact detection. Various samples exhibiting different forms of multimeric aggregation were stressed under a range of temperatures and pressures. The aggregated samples were fractionated using SEC and analyzed as the monomeric form and stressed fraction. Different aggregate samples are characterized showing only dimer (~300 kDa) in some cases and higher order (trimer [~450 kDa] and a tetramer [~600 kDa]) aggregates in other cases. Unfractionated samples of pharmaceutical formulation containing aggregates were also analyzed directly without SEC cleanup. In the mass spectrum collected, many various proteins are detected due to the absence of SEC purification, as well as the monomer and aggregated dimer peaks. To evaluate the ability of this method to relatively quantify the relative percent of aggregation, a therapeutic protein that reliably forms a dimer was utilized. The sample fraction corresponding to the monomer species was used as a baseline containing no dimer. The fraction corresponding to the dimer fraction was considered as maximum dimer signal. The collected samples (monomer and dimer) were mixed together and the percentage of dimer was plotted as the function of the area of the monomer and dimer peaks (RM and RD) creating a standard curve with an R-squared greater than 0.99. The technique described provides a tool for rapid identification of aggregation specifies as well as semi-quantitative measurement of therapeutic antibody pharmaceuticals using MALDI mass spectrometry. Results show good agreement with the orthogonal techniques however required a less sample volume and analysis time.