Teams often strive to implement appropriate analytical methods in early-phase development and minimize changes as product development progresses. To this end, platform methods have gained popularity for monoclonal antibodies (mAbs). Nevertheless, at least one analytical method inevitably evolves between FHD and commercialization. This presentation will discuss factors that drive method changes and approaches to analytical method bridging that balance the desire for exhaustive datasets with material and resource availability.

This presentation surveys the current landscape and explores innovative methods and strategies for analyzing particles in biologic drugs. It delves into recent advancements that enhance our understanding of particle characterization, origin, and the ongoing pursuit and future direction of improved analytical tools for ensuring the quality and effectiveness of biologic therapies.

Platform methods act as a great tool to support efficient and faster readiness to onboard or advance a new program through the clinical phases. It is important to have a comprehensive understanding of platform methods to build confidence in the use of these tools to support the development of new molecules. This presentation will focus on strategies to support the implementation of robust platform methods with focus on ELISA-based assays.

Antisense oligonucleotides (ASOs) are short nucleotide sequences designed antisense to target RNAs. To increase their nuclease resistance the phosphate (PO) backbone is often modified to phosphorothioate (PS), creating a new chiral center. ASOs with many PS chiral centers are mixtures of thousands of diastereomers. We compared circular dichroism, phosphate-31 NMR, and LC-MS for characterizing the diastereomeric distribution of ASOs.

This study delves into the comprehensive profiling of in vitro transcribed (IVT) mRNA impurities, crucial for enhancing safety and efficacy in biotechnological applications. Leveraging advanced analytical tools such as ion-pair reversed-phase liquid-chromatography, capillary gel-electrophoresis, microcapillary-electrophoresis, mass-photometry, and native mass-spectrometry, we unveil impurities related to mRNA variants and double-stranded mRNA byproducts. Our findings emphasize the need for improved analytical characterization, offering valuable insights for optimizing IVT mRNA production in biotechnological contexts.

Synthetic oligonucleotides have emerged as effective treatments for genetic diseases. Oligonucleotide therapeutics are commonly modified with a substitution of a phosphorothioate linkage along the phosphodiester backbone which creates a mixture of diastereomer structures. Analytical methods to measure the diastereomers are currently lacking despite recent draft guidance highlighting the importance of their characterization. Here, we present a method combining tandem MS and tandem IMS to study diastereomers in modified oligonucleotides.

To improve understanding of the structure-function relationship of a Pneumococcal conjugate vaccine, several LC-MS assays were developed for process development and characterization. Notably, Serotype 5 polysaccharide which contains a ketone group in its repeating unit is included in some vaccine formulations. An LC-MS based assay procedure paired with an isotope-labeling strategy was developed and will be presented to characterize the integrity of the ketone group after the conjugation reaction.

The identification and monitoring of residual host cell proteins (HCPs) in adeno-associated virus (AAV) by LC-MS is critical for maintaining product quality. However, applying LC-MS-based techniques to AAV poses unique challenges. In this presentation, we will explore these challenges and strategies to overcome them in the context of AAV HCP analysis. Additionally, we will present a newly developed, highly sensitive method that preserves AAV integrity while preferentially digesting HCPs.

Aggregation is a critical quality attribute that needs to be appropriately controlled in any biopharmaceutical product. For AAV-based therapeutics, methods to assess aggregation present a unique challenge. In this study, we compared strengths and weaknesses of SEC, AF4, analytical ultracentrifugation, and mass photometry. We present a case study in which very large aggregates were generated to evaluate the performance of each method in the separation and quantitation of aggregates.

mRNA vaccines offer transformative potential for disease prevention, but their rapid development demands advanced analytical techniques. This presentation delves into the latest breakthroughs in mRNA vaccine analytics. Attendees will explore cutting-edge methods for characterizing mRNA integrity, purity, and potency. Discover how these innovations optimize vaccine stability, safety, and efficacy throughout the preclinical development process.

I will present newly developed methods for characterizing AAV vector via PacBio long-read sequencing. Our tools quantitate residual impurities as well as vector subspecies (ie full vs partial) all within one assay, allowing deeper, more comprehensive understanding of vector batch content.

Our group gathers chemical liability data to inform biologics candidate design or advancement in the late Discovery pipeline. We miniaturized forced degradation by focusing on mass spectrometry because of the richness of data and broad applicability across biologic formats. The recent construction of a structured data warehouse allows broader access to data by collaborating scientists. Overall, these approaches enable comprehensive, timely, and pipeline-appropriate decisions on biologic candidates.

New Peak Detection (NPD), a critical component of Multi-Attribute Method (MAM), detects peak variations effectively. An efficient NPD method with improved sensitivity is vital for monitoring process-related attributes. This study presents the development of a robust NPD method that successfully enhances sensitivity and maintains controlled false positives. The efficacy of this innovative NPD approach was assessed at various applications to fulfill pipeline needs, exemplifying its significant potential in drug development.

Top-down and middle-down mass spectrometry (TD/MD-MS) are emerging techniques that minimize sample preparation and preserve endogenous post-translational modifications (PTMs) compared to bottom-up MS. Here, we show that assigning non-canonical internal fragments in TD-/MD-MS helps recover nearly 100% of the sequence and reveal important disulfide connectivity information of an intact mAb. In addition, drug conjugation sites can also be determined for a heterogeneous lysine-linked ADC using this novel approach.