Novel protein modalities are emerging to deliver sophisticated mechanisms of action canonical antibodies cannot. However, little is known about the in vivo stability liabilities (i.e.; biotransformation) with these new modalities. A multi-pronged approach was established using LC-MS and capillary electrophoresis-based methods to characterize and quantify biotransformation liabilities and the in vitro/ex vivo vs. in vivo translatability including but not limited to chemical linker deconjugation, clipping, and amino acid level modifications.

With 11 FDA-approved and marketed entries, antibody-drug conjugates (ADCs) have become clinically and commercially successful cancer treatments that maximize therapeutic potency and limit systemic toxicity through the selective delivery of highly potent drugs. Among many conjugation strategies, site-specific bioconjugation methods including our dual variable domain (DVD)-based technology have been developed to improve the homogeneity and stability of ADCs, aiming to advance clinical outcomes. With the growing interest in the innovation of site-specific and orthogonal bioconjugation methods for single and dual payloads, analytical characterization and functional evaluation are important to provide a baseline for their safety and efficacy profiles.

ADCs are amongst the fastest-growing drug classes in oncology evidenced by the boom of recent new approvals. In vivo PK, biotransformation, and payload tumor delivery are key information to guide linker drug design and selection. This presentation will share our bioanalytical strategy and methods to characterize novel drug conjugates from in vivo samples, and how the data is used to understand efficacy/toxicity to select molecules with optimized therapeutic index.

Antibody-drug conjugates (ADCs) have become a promising class of antitumor agents for treating cancer patients. For the ADC conjugations, traditional and site-specific approaches are being considered. To overcome the heterogeneity observed by traditional conjugations, site-specific conjugations are becoming more and more prevalent. They have been shown to eliminate heterogeneity and improve conjugate stability. This presentation will focus on the various analytical techniques used for analyzing a site-specific ADC molecule.

mRNA vaccines are a newly established class of safe and effective biotherapeutics. The mRNA is manufactured using an in vitro transcription process followed by purification, and the drug product process involves formation of mRNA-containing lipid nanoparticles. A comprehensive control strategy ensures consistent quality, and characterization studies strengthen process and product knowledge. This presentation introduces novel approaches to mRNA fragment characterization to evaluate the risk of off-target or truncated antigen translation.

RNA interference (RNAi) offers a promising therapeutic approach for the treatment of genetic diseases. Triggered by small interfering RNAs (siRNAs), RNAi silences genes by inhibiting translation of problematic transcripts. Fundamentally distinct from antibodies and small molecules, siRNAs require different strategies to evaluate purity and stability as well as support developmental and regulatory processes. Here, we show a series of analytical methods characterizing the purity and biophysical properties of therapeutic siRNAs.

While SV-AUC is revered as a gold-standard technique, it is also maligned as an outdated and cumbersome technique. This talk seeks to introduce the audience to the modern AUC platform and to highlight the unique insights offered by SV-AUC with case studies covering the characterization of AAVs, LNPs, and nucleic acid. Along the way, several myths regarding SV-AUC will be dispelled showing SV-AUC can be a medium-throughput, must-have technique!

mRNA-containing lipid nanoparticle (LNP) vaccines allow for a rapid response to seasonal and emerging viral threats given the shortened process/product development timelines. Assay development to measure CQAs like mRNA concentration and average size are rate-limiting steps. We introduce a novel automated analytical technology (commercialized) that assesses mRNA concentration in LNPs and LNP size for 96 samples in ~1 hour, using 2 µL/well and without dyes, which outperforms current methods.

• Needs/techniques/workflows to characterize novel biologics PK and biotransformation (multi-specifics, fusions, ADCs, siRNA/mRNA, CAR cells)
• Critical reagent generation to facilitate the bioanalysis ​
• ADC PK analysis: how many components to monitor, preclinical vs. clinical? Immunoassay vs. LC-MS vs. hybrid?
• ADC DAR analysis: native vs. intact vs. subunits vs. bottom up
• Nucleotide analysis:  MS vs. PCR types of analysis
  

• CQAs impacting mRNA and nanoparticle safety and efficacy
• Emerging methods and instruments
• Best practices at different stages of development
• Process analytics and QC/release testing
• Problems and solutions​

Mass spectrometry is part of the traditional toolbox for biotherapeutic characterization. For multi-specific proteins, challenges arise when new domains contain complex PTMs or necessary linkers lead to clipping events. Using electron activated dissociation, we are able to characterize clipping events for species annotation and get unambiguous assignment of glycan species using middle-down and bottom-up analysis, respectively, for a more in-depth understanding of our therapeutic candidates.

Antibodies in solution are inherently dynamic molecules stability that can present unique challenges for the development of stable liquid formulations. Their stability profiles and quality attributes are often linked to fluctuations in intramolecular conformations and structural flexibility. HS-AFM provides information on structure and dynamics at the single-molecule level, thus potentially advancing our understanding of the inter-relationship between the nanoscopic observation of molecular behavior and the macroscopic stability of biopharmaceutical formulations.

mRNA vaccines are currently at the forefront of the vaccine industry due to their safety, efficacy, and fast turnaround times between pathogen detection and vaccine development. However, proper analytical methods to assess the purity of these samples are still lagging. Here, we propose a tool that can streamline the characterization of mRNA vaccine purity, with a focus on dsRNA contaminants, through the use of microfluidic electrophoresis and differential labeling. In addition, with its high-throughput compatibility and short analytical time per sample, this method can has great potential for batch-to-batch analysis.

Surface plasmon resonance (SPR) has been widely used in biotherapeutic development for decades. However, no systematic study has been performed on how to qualify SPR assays for the various SPR result types need for regulatory documents. This talk will discuss methods and guidelines for SPR assay qualification in various scenarios. We hope our studies can help align SPR applications among the scientific communities involved in drug development.

In recent years, a great percentage of biotechnology and biopharma companies' portfolios have comprised novel protein and antibody-based therapeutics. Developing and optimizing these molecules requires detailed analytical characterization. In our current work, we utilize liquid chromatography coupled with intact or top-down mass spectrometry in order to understand the biotransformation of novel modalities (trimeric Fab, antibody-drug conjugates, VHH), which facilitates the interpretation of in vivo findings and (re-)engineering of more stable molecules.

Protein-based therapies hold an enormous potential for treating many terminal diseases. Nevertheless, the lack of universal technological approaches that enable development of protein formulations with the targeted attributes significantly impedes clinical translation of these advanced therapies. This presentation will overview the use of droplet based microfluidic technology for developing protein formulations with pre-programmed functional characteristics, including size and internal morphology, encapsulation efficiency, and protein release profile.