There has been much recent interest in protein drug conjugates, especially antibody drug conjugates, as an important pharmaceutical class of drugs in targeted therapies which take advantage of the high specificity of antibodies and the potency of small molecule therapeutics. These complex molecules, though, present several analytical challenges including the determination of the drug-to-protein ratio and characterization of conjugation sites. Here we outline some of our experiences using various mass spectrometry-based tools and strategies. The presentation will discuss specific conjugation chemistries used in our work, determination of drug-to-protein ratios and conjugation stoichiometries, and characterization of conjugation sites?.

The talk will describe LC-MS methods used to characterize degradation products of ADCs. More specifically, mass spectrometry techniques were used to identify and quantify site-specific hydrolysis on the linker and payload. Furthermore, an LC-MS method for determination of the global payload hydrolysis levels was established to enable a more informed control strategy.

Antibody-drug conjugates (ADCs) have become an attractive modality for treating complex diseases. In vivo, ADCs could undergo biotransformation, leading to modifications of the linker-payload and/or changes in the drug-to-antibody ratio (DAR). These modifications could affect efficacy or introduce toxicity. Investigating the in vitro/in vivo stability/biotransformation could provide guidance to linker-payload and antibody selections. In this work, case studies of how biotransformation data informed lead candidate(s) selection or optimization are presented.

Here we present two CE-western assays for quantifying the relative stoichiometry of VP, and residual levels of a baculovirus protein impurity, GP64. Various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results were further supported by LC-MS analyses.

There is a high demand to define analytical strategies for the characterization of adeno-associated virus (AAV)- and lentivirus (LV)-based drug products, because it is essential for ensuring safety and efficacy. In this context, both the characterization of the virus itself and the analysis of particulate impurities are crucial. While for AAV, already many biophysical methods to characterize the virus itself have been developed, fewer methods are available for LV. Using a comprehensive set of particle analysis techniques allows us to generate a comprehensive understanding of particulate impurities present in both AAV and LV drug products.

Antibody-siRNA conjugate (AOC) has the potential to overcome the obstacles of systemic delivery of siRNAs. To cross the blood-brain barrier (BBB) for siRNA delivery, the antibody must have the affinity to the receptor to allow for brain uptake and subsequently be released for neuronal uptake. We have developed a set of tools to screen for antibodies with such an ideal range of affinity and to assess the stability of the AOCs.

Ensuring the efficacy of mRNA therapeutics requires precise characterization and control of critical quality attributes at both the mRNA and lipid nanoparticle (LNP) levels. This talk explores advanced analytical methods, with a focus on mass spectrometry, to assess essential mRNA attributes-sequence fidelity, purity, cap/tail integrity, and process-related impurities-highlighting orthogonal approaches that support the structural and functional integrity of mRNA therapeutics.

• Key challenges and strategies for analyzing particulate impurities in gene therapy products, such viral vectors, mRNA-LNPs, etc.
• What types and sizes of particles should we expect?
• How do we assess impurities, when the active itself is a particle?
• What are key techniques for sizing, counting, characterization, and identification or particles?
• Are particulate impurities cQAs for gene therapy products, as in biologics?

This talk explores the multifaceted approach of combining antibody and oligonucleotide modalities, using Brainshuttle-ASO as a case study for delivering antisense oligonucleotides (ASOs) to the brain. We will discuss the design process, developability, and molecule stability. Closing with in vivo activity data, a holistic view of the journey from concept to practical application will be provided.

Functionalization of therapeutic oligonucleotides by conjugating to multiple modalities may be beneficial for their efficacy or delivery. Certain examples like GalNAc have been widely applied in therapeutic ASO and siRNA development. Maleimide-conjugated ASO can be easily functionalized by conjugating to thiol-bearing modalities. During the manufacturing development of such conjugates, we have encountered several difficulties, conquered them, and learned lessons. This presentation intends to share what we learned thus far.

As emerging therapeutic and vaccine platforms, oligonucleotide-based modalities have shown great success in treating diseases and in COVID pandemic response. Defining and measuring the quality attributes of oligonucleotide-based modalities presents new analytical challenges. To this end, I will describe new measurement technologies and reference materials being developed at NIST to support analytical characterization and harmonization of methods for oligonucleotide therapeutics, from short siRNA and antisense drugs to mRNA vaccines.

Bispecific antibodies (bsAbs) have emerged as a novel therapeutic antibody format, leveraging two distinct Fab domains to directly bind multiple targets. Although this format enables bsAbs to address complex diseases, each Fab arm is differentially prone to modification, adding complexity to the charge profile. Here we present an effective workflow to characterize charge heterogeneity in bsAbs, using platform-based enrichment followed by domain-specific charge variant analysis, identity, and potency characterization.

Recombinant polyclonal antibodies (pAb), a new class of therapeutics comprising over 1000 immunoglobulin G1 (IgG) species recognizing multiple epitopes or antigens, are anticipated to be highly effective for treating rapidly evolving infectious diseases. We developed a CEX-HPLC method to address a major hurdle in pAb manufacturing, the reliable characterization of pAb population diversity and identity. The method generates a reproducible and unique profile for a pAb product, is sensitive to lot-to-lot variation in pAb population and can serve as a fingerprint identity assay.

A review of methods for isolating and characterizing EVs in order to address the challenges associated with the widespread heterogeneity in the size and composition of EVs. We will present a high-throughput single molecule detection array (SiMoA) for fast immune-phenotyping of extracellular vesicles from low-volume biofluids. The array has been used to observe changes in protein expression across different EV populations and identify key proteins associated with disease progression.