RESUMEN
The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.
RESUMEN
Mild or elevated temperatures are routinely used for the analysis of therapeutic proteins by reversed phase liquid chromatography. Generic conditions can be used for the analysis of monoclonal antibodies, and may be adapted for species derived thereof, for instance their immuno-conjugates. Beyond platform monoclonal antibodies, many novel, non-covalent protein complexes are also frequently pursued as protein therapeutics. These complexes, in reverse phased chromatography, may require extremely harsh, superheated conditions to dissociate and elute as interpretable profiles. In order to minimize on-column degradation under superheated conditions, the analysis time has to be reduced as much as possible. Using ultrashort columns and fast gradients is a promising approach in achieving informative separations within a minute, or even faster. Here the applicability of this approach, which supports maintaining levels of degradation products close to the intrinsic sample composition without further on-column degradation is demonstrated. NISTmAb as conventional IgG, a bispecific homodimer and a bispecific homotetramer were used for demonstrating differences in the elution characteristics and the necessity of using the proposed approach. The analysis of the bispecific homodimer was discussed in detail as a case study.