Molecular interaction characterization (MIC) of therapeutic antibodies binding to their targets is critical at each stage of antibody therapeutic development. The introduction of new antibody modalities poses challenges to the MIC assay development due to their complex structural and molecular properties, and requires fit-for-purpose MIC assay strategies. This presentation will focus on case studies that demonstrate how fit-for-purpose MIC strategies are applied to overcome these challenges to enable IND filings.

Clinical use of the interleukin-2 (IL-2) cytokine has been limited by its pleiotropic immune activities as well as its short serum half-life. Recently developed cytokine/antibody fusion proteins (immunocytokines) that link IL-2 to biasing anti-IL-2 antibodies help to overcome challenges for IL-2 therapies. We build on this advance to target and retain immunocytokines within the tumor microenvironment. We characterize different fusion topologies and test multiple animal models using various routes of injection to identify the optimal formulation for our engineered immunocytokine.

Understanding the structure of mRNA lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is fundamental to advancing their biomedical potential. Elucidating this detail via direct experimental methods has been a major objective in the field of RNA delivery. Here, we show that a combination of dye-binding and cryo-electron microscopy pinpoints the mRNA location, providing new insights into its encapsulation state and chemical microenvironment.

Multispecific antibody formats which employ multiple distinct chains and asymmetric pairing are especially challenging analytically due to the potential for chain mispairing. Characterization of these molecules by liquid chromatography-mass spectrometry (LC-MS) and analytical hydrophobic interaction chromatography (aHIC) enables identification and quantification of the molecules, respectively. This presentation highlights the insights revealed using a combination of these techniques, and how the data is used to guide early-stage research decisions.

Peptide mapping based Multi-Attribute Method (MAM) is attracting many industry users as it allows deep characterization of biopharmaceuticals in a QC environment. We established an intact Multi-Attribute Method (iMAM) for a streamlined and automated processing of MS data acquired at intact monoclonal antibody level. Using three case studies, method robustness, accuracy and reproducibility were evaluated for correct protein ID confirmation, proteoforms quantitation and identification of new entities.

Polyclonal antibodies are popular reagents for their high sensitivity and robust cross-platform performance, but suffer reproducibility challenges and limited supply. Breakthrough polyclonal sequencing technology can overcome these limitations by capturing the most abundant IgG sequences and enabling indefinite recombinant antibody production. Here we report the first successful conversion of a [nepalese] goat polyclonal antibody into recombinant mAb cocktail using only the pAb protein sample.

As the number of biotherapeutic candidates is increasing, the requirements of analytical tools to understand these molecules are rising. To understand product quality attributes (PQAs) fully, identification, localization, and relative quantification are key. Here, a novel mass spectrometry (MS) fragmentation technique was evaluated for peptide mapping: electron activated dissociation (EAD), that creates MS/MS fragment ions of the peptide backbone while maintaining intact PTMs on the fragments.

Utilizing capillary gel electrophoresis (CGE) and capillary zone electrophoresis (CZE), PerkinElmer’s LabChip GXII Touch Protein Characterization System provides characterization of proteins and nucleic acids in as little as 42 seconds per sample, delivering data comparable to other methods of quantitation with as much as 70x increase in throughput. The system can quickly measure yield, purity, titer, charge variation, and glycosylation of proteins.

Nonspecific binding is a common phenomenon that plagues many antibody discovery and optimization campaigns. The ability to rapidly identify and reduce off-target binding early during candidate selection can help improve the odds of selecting lead antibodies with drug-like properties. I will discuss trends in antibody developability from our analysis of clinical and pipeline datasets across antibody modalities, and apply these learnings with a recent case study that utilizes predictive library design and high-throughput developability screening to improve specificity during optimization.

Protein developability challenges hinder the discovery and engineering pipeline. We present three high-throughput genotype-phenotype-linked assays that measure developability proxies for 105 variants (with efficient scalability to 107). A random forest model trained on the assay data predicted recombinant expression of scaffold variants with 5x greater efficiency and 35% increased accuracy than a sequence-derived model. These assays enable developable protein discovery, inform protein design, and allow more precise landscape visualization.

Mass spectrometry is a powerful analytical technique in the identification, characterization, and quantitation of biologics. More recently, structural mass spectrometry methods have shown tremendous potential in understanding higher-order structures of biologics. In this presentation, we demonstrate how product characterization can benefit from structural mass spectrometry approaches. Native SEC-MS and Native-MS coupled with ion mobility can be used to rapidly screen molecules for potential aggregation risks while HD exchange, crosslinking and footprinting methods allow more in-depth structural information, i.e., binding interface, epitope, and paratope mapping. Emerging structural MS technologies will be presented in the context of discovery and early drug development of biotherapeutics

Lipid nanoparticles (LNPs) represent a promising platform for the intracellular delivery of biological therapeutics and vaccines. Molecular assembly of this lipid-based drug delivery system significantly impact the stability and drug release properties of the nanoparticles. However, the structural basis of LNP assembly and encapsulation for drug product development is largely unexplored. Our study aims to understand the interplay of formulation processing and composition parameters and their impact on LNPs formation and RNA encapsulation by elucidating high-resolution structural details.

Large molecule characterization assays can be cumbersome and difficult, largely due to inefficiencies in liquid-phase separation required for resolution of critical peptide modifications. High resolution ion mobility (HRIM) provides gas-phase separation of ions that rivals the most efficient liquid phase separations on a millisecond timescale. This seminar will focus on HRIM applications to current-generation LCMS assays and the analytical gains the system provides in identifying and monitoring critical quality attributes.

This presentation will discuss Key Concepts and Challenges in Freezing PROTEINS: Ice-water Interface & Cold Denaturation

Thorough characterization of both reagent and therapeutic protein candidates through use of various analytical methods is critical to the success of a therapeutic drug discovery program. Here we highlight the utility, challenges and limitations of both native and denaturing mass spectrometry to characterize a variety of difficult proteins such as the heavily glycosylated SARS COVID-19 spike protein, various peptide-MHC complexes and a heavily clipped monovalent bispecific fusion antibody.

In this presentation, we will discuss single-cell analysis to characterize the CHO cells.

There is a growing need in biologics manufacturing for process analytical technologies (PAT) with the ability to accurately test protein product ID quantitation and higher-order structure assessment. We present Culpeo® a non-destructive QCL-IR liquid analyzer that provides protein ID testing, simultaneous protein quantitation, and secondary structure analysis in either in-line or at-line workflows. We show the specific workflow for using Culpeo®  as an orthogonal tool in protein ID testing and quantitation.

Understanding the interactions of viral proteins with receptors and other binding partners is key to successful development. This presentation will cover the theory of light scattering and how size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and composition-gradient light scattering (CG-MALS) are used for biophysical characterization and determination of binding affinities highlighting case studies of various proteins and oligonucleotides.