“Separation of Monoclonal Antibody Monomer from High Molecular Weight (HMW), Low Molecular Weight (LMW) and Other Heterogenic Impurities using Different Modes of Chromatography” - Atis Chakrabarti, Ph.D.
Protein aggregation is a biological phenomenon. There are many reasons why the aggregates or the high molecular weight (HMW) species form. Mostly the mis-folded proteins have a tendency to form aggregate. Both intra- or extra-cellular protein aggregates are found. These protein aggregates are often toxic; protein aggregates have been implicated in a wide variety of disease known as amyloidosis, including Alzheimer’s, Parkinson’s and prion disease.
Nowadays, monoclonal antibody proteins are widely being used in the field of bio therapeutics. These bio therapeutic proteins must be free from aggregates. Separation of the pure antibody monomer needs to be very well resolved from its dimer and higher order aggregates. Similarly for quality control and regulatory purpose the separation of antibody fragments or low molecular weight (LMW) species is also very much essential.
The species other than the monomer might induce toxic side effects to the body if not removed. Even if the impurities are non-toxic, the mere presence of these species may reduce the potency of the final formulation and hence need to be removed.
Many chromatographic modes can be coupled to separate complex mixtures, variants or impurities otherwise inseparable by a single mode of chromatography. A very favorable combination is Size Exclusion Chromatography (SEC) and Reversed Phased Chromatography (RPC). SEC and RPC are some of the most frequently used chromatographic modes for analytical separations of biomolecules, particularly monoclonal antibodies. The separation mechanism is orthogonal and the mobile phases are compatible.
Analytical SEC columns particularly one packed with 30 nm wider pore size, 3 μm particles with larger molecular exclusion limit can be used to separate as well as quantitate the aggregates or HMW species as a function of their hydrodynamic volume. A well resolved symmetric and apparently pure protein peak obtained from size exclusion chromatography need to be checked by another mode for the presence of any variants. A reversed phase chromatography using analytical column with wide pore size of 30 nm for maximum mass transfer is expected to be useful for the separation of large biomolecules such as proteins as well as their hydrophobic variants.
Here we discuss the use of analytical columns from two different modes of chromatography (SEC and RPC) in separation of monomer of proteins and monoclonal antibodies from their HMW and LMW impurities and variants.
“Light Scattering for Absolute Macromolecular Characterization” - Stephanie Cope, Ph.D., Applications Scientist, Wyatt Technology
Light scattering (LS), including classical and dynamic, has been widely employed to characterize protein solutions and other biomolecules. Static light scattering, especially multi-angle light scattering (MALS), determines the absolute molar mass, size, and conformation of macromolecules in solution, facilitating the characterization of molecular heterogeneity and impurity profile. Dynamic light scattering (DLS), also known as quasi-elastic light scattering (QELS), directly measures the translational diffusion coefficient from which the hydrodynamic radius of a molecule is derived. The power of MALS/QELS combined with HPLC (such as SEC, RPC, IEC, etc.) will be discussed and demonstrated through various applications.