Purpose: To differentiate MabSelect SuRe LX (MSS-LX) sepharose from other resins, especially very similar protein-based sepharose subtypes, tandem identification methods, Near Infrared (NIR) and visible spectroscopies, plus protein-binding capacity (PBC) methods, have been developed and validated.
Methods: For NIR and visible spectral identification method, reference spectra from multiple lots for each of 16 resin analytes were collected to build a library system. The library configuration and parameters were optimized to include first-tier global identification and second-tier local qualifications. Various spectral pretreatments and chemometric methods were investigated to maximize identification ability in each tier of the library system.
Additional identification methods, such as amino acid analysis (AAA) and PBC using variable path extension (VPE) spectrometry, have been developed to distinguish MSS-LX from MabSelect SuRe (MSS) since both were still ambiguous with respect to each other in NIR identifications.
During method validation, negative challenge resins, positive control samples, and robustness experiments were examined.
Results: A global library identification (1st-tier spectral library) used three wavelength segments and spectral pretreatments to exclude moisture absorption bands and to minimize the impact from sample preparation and environmental variations. Twelve of 16 resin analytes reached clear identifications. Ambiguous MSS-LX, MSS, rProtein A (rPA) and MabSelect sepharoses were then searched through 2nd-tier local libraries. As the spectra of MSS-LX and MSS are near identical throughout the entire scanned region (400-2500 nm), local qualification steps only eliminate their interference for identifications of rPA and MabSelect, which were conclusively identified. Over 30 additional negative challenge resin species were examined. Long term variability had no adverse effect on identifications.
The PBC method is superior to the AAA method for distinguishing MSS-LX from MSS. For PBC method, ratio and amounts between protein substitute and resin (MSS-LX or MSS), binding kinetics and identification threshold were optimized and validated.
Conclusion: This NIR and visible spectroscopic method combined with protein binding capacity method is selective, robust and reproducible to identify MabSelect SuRe LX sepharose and 15 other resins.