To support the fast-to-release paradigm for next-generation molecules such as trispecifics, it’s important to develop high throughput, robust assays that support releasing clinical materials in a quality control laboratory. This presentation will detail the development of relative potency binding ELISAs utilizing Meso Scale Discovery’s (MSD) electro-chemiluminescence U-PLEX platform technology for trispecific antibodies, thereby generating relative potency results for each arm of the trispecifc in one assay occasion.

ADC bioanalysis strategies are shared for pre-clinical ADC studies. Total antibody and total ADC quantitation by digestion approaches are accompanied by free payload quantitation for determination of PK concentration, efficacy, and safety. Intact mass analyses of ADCs from in-life studies are also leveraged to calculate in vivo DAR. Presented here are current MS methods for pre-clinical ADC bioanalytical support, including hybrid immunocapture MS formats and payload quantitation.

DNase I digestion is commonly used to remove residual plasmid DNA (pDNA) from mRNA drug substances, yet its optimal conditions and enzymatic behavior remain underexplored. We developed two RP-IP chromatography methods to characterize DNase I digestion with high sensitivity and single-nucleotide resolution. These methods complement qPCR by providing structural insights into DNA fragments and confirm effective pDNA removal in the final drug substance under controlled digestion conditions.

The Intact Multi-Attribute Method (iMAM) is a powerful analytical approach for characterizing and ensuring the quality of therapeutic antibody-oligonucleotide conjugates (ARCs). This presentation explores the implementation of iMAM in biopharmaceutical development, highlighting its ability to provide detailed structural insights, monitor product consistency, and support quality control. By integrating high-resolution mass spectrometry, iMAM enhances the detection of critical quality attributes, offering a robust alternative to traditional assays for ARC characterization.

Mass spectrometry (MS) plays a crucial role in the characterization of glycoproteins, providing detailed insights into glycosylation patterns, site occupancy, and structural heterogeneity. In biologic drug development, MS enables high-resolution analysis of glycan structures, essential for ensuring product consistency, efficacy, and safety. This presentation will explore advanced MS techniques, including glycopeptide mapping and intact glycoprotein analysis, highlighting their applications in regulatory compliance and quality control of therapeutic glycoproteins.

Predicting protein aggregation remains difficult, limiting their use for biotechnology and therapeutic applications. We aim to design aggregation-resistance by collecting and learning from large, experimentally validated datasets. I quantified aggregation after thermal and pH stress for thousands of small protein domains using mass spectrometry. I’m developing machine learning models to predict aggregation from protein features. Through iterative experiments and design, I will refine my model to achieve unprecedented aggregation-resistance.

Dynamic products like respiratory vaccines often require identity method updates during seasonal variant/strain changes. For a ddPCR-based method, this involves redesigning primers and probes for both the previous season and the upcoming strains to maintain specificity. This task becomes even more challenging when dealing with multivalent vaccines, where the ratio of nucleic acid molecules is as crucial as their identity. This talk will cover the development of a platform next-generation sequencing ID/ratio method, from early data showing proof of concept work and the path to GMP for all instrumentation and bioinformatics, culminating in the completion of method qualification.

Phosphoramidites are the building blocks currently used to manufacture therapeutic oligonucleotides. USP has been exploring new opportunities to develop standards for phosphoramidite, including DNA, RNA, MOE amidites, and impurities amidites. A multi-center collaborative study extensively characterized USP standards, suitable for use as reference standards for raw materials in oligonucleotide-related products.

In this contribution we show how native mass spectrometry in the Megadalton range can be used to accurately measure masses of recombinant AAV particles of varying compositions. We discuss approaches to increase mass resolving power and deconvolution of signals on commercial instruments such as the Thermo Scientific Orbitrap Exactive UHMR using charge detection and charge-shifting solution additives. We are also discussing characterization of AAV topology and cargo release.

rAAV genomes comprises a heterogeneous population within AAV particles. These particles contain the intact genome but also include numerous truncated species, which likely lack functionality and may induce adverse effects. Viral genome titer methods do not accurately reflect this heterogeneity and there are no reliable quantitative methods. We have developed a novel high-throughput RNA-DNA hybrid method for quantitating the intact and truncated rAAV genomes. A much-needed advancement for the field.

• What methods does industry use in general to evaluate the multispecific molecule? 
• Is efficiency for release testing a consideration?
• Regulatory feedback on strategy employed to evaluate potency of multispecifics; dual binding approach for bispecifics feedback discussions 
• Regulatory expectations around cell-based vs binding potency: Has any company managed to replace the cell-based w/binding for novel modalities?​

• What testing of ADC reference materials helps ensure meaningful outcomes from early in-life studies?
• Role of serum stability for ADCs
• First dosed-study: In-life stability, in-life PK, or both?
• Thresholds and characterizations of free payload in early discovery ADC reference material​

Full-scale molecular dynamics simulations of viral capsids provide a well-established platform for high spatial and temporal resolution analysis of capsid mechanoelastic properties. These simulations provide accurate molecular views that are successfully validated against multiple experimental techniques. Altogether, our results suggest that the HIV-1 capsid is a robust container with finely tuned viscoelastic properties that allow it to adapt to a range of geometries during cytoplasmic and nuclear trafficking.

rAAV vectors can be produced in various cell types, including the baculovirus/Sf9 system, which introduce residual DNA impurities. We characterized and quantified baculoviral (BV) DNA and determine the mechanism for introduction of these impurities. This demonstrates important features of the BV plasmid design including antibiotic resistance gene location, questions the need for stuffer DNA, indicates selective packaging, and highlights important aspects for design of residual BV DNA quantitative methods.

In CRISPR/Cas9 genome editing system, phosphorothioate (PS) modifications in single-guide RNA (sgRNA) introduce chiral centers, creating complex isomers. This study presents a novel approach using endonuclease digestion and ion pairing reversed-phase liquid chromatography with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to differentiate and quantify PS-induced isomers. This method, rigorously evaluated, also investigates the conversion kinetics from PS to phosphodiester (PO) impurities under oxidative stress, offering insights into PS stability.

Non-specific interactions are a key developability parameter to monitor during monoclonal antibody discovery and development. However, the underlying physicochemical parameters remain poorly understood, limiting our ability to identify candidates with a propensity for non-specificity. Here, we employ microfluidic technologies to generate non-specificity fingerprints for a panel of clinical-stage antibodies in solution, providing quantitative data on the underlying physical chemistry. Based on our findings, we propose a quantitative non-specificity score, which can be integrated in the development workflow for biological therapeutics and more widely in protein engineering.

Dielectrophoresis (DEP) is a valuable technique for single-cell analysis in a population of mammalian cells. Using a selected frequency, viable and non-viable cells can be distinguished by their differential trajectory in a microfluidic cytometer. DEP analysis can also be used to measure changes in cytoplasmic conductivity or changes in membrane structure. DEP is a label-free technique and can identify early changes in metabolism that occur toward the end of bioprocesses. In particular, early detection of apoptosis allows intervention to reverse the pathway to cell death or to terminate the culture prior to the excessive release of host-cell proteins (HCP).

This presentation introduces a tiered, high-throughput analytical workflow designed to optimize Cell CLD by balancing speed and depth in early-stage screening. It will cover the integration of high-throughput assays, automated purification, and in-depth characterizations to efficiently evaluate pools and clones while ensuring product identity and integrity. Emphasis will be placed on overcoming challenges with complex formats such as bispecific antibodies and streamlining clone selection based on critical quality attributes (CQAs).