Two-dimensional chromatography represents the most thorough and rigorous approach to evaluation of the proteome. Variants can readily be identified and lot comparisons made by combining second- dimension chromatography with DC as the first dimension. In our case, the displacement fractions are run neat or diluted in DI water. The reverse-phase solvents are by convention installed on the HPLC channels A and B. The A solvent by convention is the aqueous solvent (water) and the B solvent by convention is the organic solvent (acetonitrile, methanol, propanol). We used a XBridge C18 column (Waters) 250 x 4.6 mm at 1 mL/min flow rate with a gradient from 10% B to 30% B over 40 min followed by a gradient from 30–100% B over 20 minutes.
Figure 2 shows the two dimensional results from the DC run in Figure 1 that have been chromatographed in a second dimension using subsequent elution separation as the chromatography mode. In this 2-D chromatogram, the location of the acidic variant is noted as well as the main antibody peak and basic variants. These peaks may be further isolated and evaluated by or, in the case of secondary reversed phase elution chromatography, may be run directly on-line to a mass spectrometer.
Product heterogeneity is common to monoclonal antibodies and other recombinant biological production and is introduced either upstream during expression or downstream during manufacturing. These variations in the target product are known to affect protein stability and hence, product efficacy. DC approaches enable the investigator to identify and characterize these often previously unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pK studies.
Knowledge gained during the preclinical development phase is critical for enhanced understanding of product quality and provides a basis for risk management and increased regulatory flexibility. The QbD initiative attempts to provide guidance on development and to facilitate design of products and processes that maximize efficacy and safety profile while enhancing product manufacturability.
DC can therefore become an enabling technology to compare process variation from one manufacturing step to the next. It allows for the preparative separation and testing of variants that could be present issues in the clinical setting. This information would be essential in development of an adequate QbD program.