Orthogonal PAT Analytics – Essential to Enable Robust Production of Viral Vectors and LNPs

Discover the innovative presentation by Aleš Štrancar, Managing Director at BIA Separations, Inc., presented at Cell & Gene Therapy International EU in December 2025. This important talk highlights the importance of CMC and orthogonal analytics to develop and manufacture your targeted LNP formulations (tLNPs). It focuses on using PATfix LNP Switcher to monitor a crucial conjugation reaction between an LNP and a targeting ligand.

Abstract:

Lipid nanoparticles (LNPs) represent the forefront of in vivo drug delivery for nucleic acids, yet transitioning from development to commercial biopharmaceuticals presents challenges in biology, manufacturing, and characterization. The specific challenge of biodistribution and organ targeting is key for developing therapeutics like in vivo CAR-T. Targeting can be exploited endogenously or by creating active targeted LNPs with surface ligands, called targeted LNPs (tLNPs).

Such tLNP formulations are much more complex than the classic 4-lipid component LNP formulations. Thus, CMC is a major hurdle to successful therapeutic development. Beyond the common intricacies of LNP characterization, including encapsulation efficiency, particle size, heterogeneity, surface charge, morphology, and RNA loading, tLNPs require careful monitoring of the reaction with the targeting ligand and separation of particles on a particle level.

Here, Aleš focuses on using a 2D chromatographic system, PATfix LNP Switcher, to monitor an on-particle conjugation reaction for synthesizing tLNPs. The use of two monolithic columns – OH and SDVB – and three detectors – UV, MALS, and fluorescence (monitoring intrinsic tryptophan fluorescence of proteins) – enables robust monitoring of the kinetics of the conjugation reaction by observing the decrease in free protein concentration and increased on-particle protein concentration. At the same time, particle size, encapsulation efficiency, and nucleic acid integrity are measured. Additionally, IEX chromatographic methods were developed to separate, on a particle level, particles with no surface ligands from targeted LNPs, a key innovative method for intact particle separation.

Beyond the tLNP analytics, Aleš focuses on the broader scope of the importance of good orthogonal analytics in process development. The process is only as good as your analytics. The more defined the process, the easier its scale-up. To enable batch-to-batch product manufacturing consistency, orthogonal analytical methods are required; PATfix tools to manufacture AAV, pDNA, mRNA, and LNPs are showcased.