Coupling of multivariate curve resolution-alternating least squares and mechanistic hard models to investigate antibody purification from human plasma using ion exchange chromatography.

A two steps proposal for the purification of immunoglobulin G from human blood plasma is investigated. The first step is precipitation using cold ethanol based on the Cohn method with some modification and the second step is a chromatographic separation by DEAE-Sepharose FF resin as a weak anion exchanger. The presence of interferent in the region3 of chromatographic fractions, which is co-eluted with IgG, restricts the application of the mechanistic chromatography model. Therefore, multivariate cure resolution-alternating least squares (MCR-ALS) as a soft method is employed on measured absorbance data matrix from eluted fractions to recover pure concentration and spectral profiles. Besides, possible solutions for resolved concentration and spectral profiles are investigated. The reaction-dispersive model as a mechanistic hard model for the column is utilized for the evaluation of the ion exchange chromatography. Using a genetic algorithm as a global optimization method, mobile phase modulator (MPM) adsorption model parameters such as ß, kdes,0, and Keq,0, were fitted to the concentration profiles from MCR-ALS as 1.96, 2.87×10-4 m3 mol-1s-1, and 1883, respectively. Furthermore, a new resampling incorporated non-parametric statistics is conducted to assess parameters' uncertainty. Values of 2.00, 1.10×10-3 m3 mol-1s-1, and 549.80 are estimated median, and values of 0.05, 2.5×10-3, and 691.00 are calculated interquartile range (IQR) for ß, kdes,0, and Keq,0, respectively. Finally, results show three and two outliers for ß and kdes,0, respectively.

Antibodies purification from human plasma using fractionation, chromatography and gel electrophoresis assisted by multivariate analysis of complimentary absorption and fluorescence spectra.

Employing simple precipitation (fractionation) using Cohn method and weak anion exchange chromatography with DEAE resin, antibodies such as Immunoglobulin G are purified from human plasma. Fractions are eluted from column in four different regions depending on washing NaCl concentrations. Absorbance and excitation-emission fluorescence spectral data are measured for separated chromatographic fractions and analyzed using Multivariate Curve Resolution- Alternating Least Squares (MCR-ALS) and Parallel Factor Analysis (PARAFAC) techniques. Resolved concentration and spectral profiles provided information about existing components in each fraction. Protein and non-protein components are distinguished considering their resolved pure spectra and information from the two applied spectroscopic techniques is complementary. A number of components displayed both fluorescence and absorbance signals. When concentration of component (protein or non-protein) in sample is low and no significant absorbance signal is observed, sensitive fluorescence is useful to recognize the component and for non-fluorescent components absorbance spectra are utilized. Electrophoresis is utilized for separation of proteins in each fraction and showed that one distinguished protein from fluorescence and/or absorbance data can be a group of proteins with similar pure spectra and retention volume. Results showed presence of two protein in the first region (IgM and IgA), a group of proteins in second region (IgM, α-globulin, and IgG), a pure protein in third region (IgG), and a group of ß-globulin proteins in fifth region. It is well and clearly shown that multivariate analysis of different data sets with complementary information is necessary for better interpretation of such technically simple and biochemically complicated systems.