The development and manufacture of heterodimeric proteins has been hindered by the lack of readily available analytical methods to distinguish homodimer impurities from the target heterodimeric therapeutic molecule. A sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) method has been developed to separate structurally similar molecules based on their level of N-linked glycosylation. The method is utilized to support process development decisions to enrich for the heterodimeric protein, which has been confirmed through a dual binding assay.

Charge detection-mass spectrometry (CD-MS) directly measures the charge and mass-to-charge ratio, generating the molecular weight distribution for highly heterogeneous biomolecules that cannot be determined by conventional MS. Here we applied CD-MS to characterize antibody-drug conjugates, cytokine fusion proteins, and the disulfide-constrained peptides from biomatrices. Combined with bottom-up MS, CE, and HIC-UV, the CD-MS studies allowed us to perform the bioanalyses of the emerging modalities to understand their pharmacokinetics and efficacy.

ABBV-400 is an ADC consisting of the anti-cMet antibody, telisotuzumab, and a topoisomerase-1 inhibitor (Top-1i). During the development of this molecule in vitro immunosafety studies were performed. To this end ABBV-400 was evaluated for FcgR and Fc neonatal binding, ADCC-CD16 signaling, and PBC activation by means of cytokine release, CD69 expression and proliferation. This presentation will highlight the results of those studies.

AbbVie is extending the use of Recombinant Adeno-Associated Virus (rAAV) to deliver DNA encoding antibody-based biotherapeutics to target a wide range of proteins involved in neurological and eye diseases. Using engineered capsids for the delivery of the rAAV genome, we explore their manufacturability properties in comparison to the natural AAV serotypes. Insights into the production efficiency, thermal stability, and full-length rAAV genome and DNA contaminant packaging will be discussed.

Identification and further characterization of antibody size and charge variants is a crucial step during biopharmaceutical drug development of novel antibody formats. At Roche Pharma Research and Technical Development, native mass spectrometry and multi-dimensional liquid chromatography is routinely used for the characterization of biologics. This presentation will summarize the strategy and challenges that were encountered during method development of these assays.

VivoVec, a novel off-the-shelf surface-engineered lentiviral vector platform, is designed to achieve specific and efficient in vivo T cell transduction following direct administration. The VivoVec manufacturing process control and product release strategy employs methods to measure the identity, purity, potency strength, and safety attributes. Characterization of VivoVec process impurities, product attributes, and transduced T cells will be described.

In recent years, investigational CRISPR therapies either delivered by LNP in vivo or as cell therapy ex vivo have shown control of target gene expression in humans, demonstrating the modalities’ potential therapeutic capabilities. Development of these therapeutics poses a challenge for tracking these components’ pharmacokinetics, biodistribution and the resulting pharmacodynamics. Oligonucleotide bioanalysis using droplet digital PCR is an emerging technology and is becoming the preferred method for several applications due to its superb sensitivity, accuracy, and precision. Here, we discuss current and emerging applications of droplet digital PCR in CRISPR gene therapy development.

• Which specific challenges are seen when screening for developability of bispecific antibodies?
• How can the characterization of the parent antibodies inform on the properties of the bispecific antibody?
• How to assess and define purity of multivalent-antibody or multi-specific formats?
• Can a developability profile and/or formulation stability on par with standard antibodies normally be reached for multivalent or multi-specific formats?​

An increasing number of bispecific antibodies is entering clinical trials. However, bispecific antibodies add complexity to variant screening and characterization. Here, I will give an overview of techniques we use to study the developability, purity profiles, and antibody-target complexes. I will provide case stories including application of mass photometry to study antibody-antigen complexes and examples of how developability profiles of different parent mAbs translate to the final bispecific format.

Identifying drug candidates from antibody libraries requires careful assessment of their binding properties. The appropriate application of different biosensing technologies at each stage of antibody discovery and development can aid in advancing or screening out candidates. Here, I will discuss how Dragonfly Therapeutics uses a Carterra LSA, Biacore 8K, and KinExA 4000 to characterize IgG-based multispecific antibodies, from discovery through lead characterization.

Bispecific antibodies are capable of binding to two antigens or two domains of the target antigen(s). This presentation will briefly discuss the analytical development and characterization strategies that are applied to elucidate the structure of bispecific antibody molecules and to better understand the potential impurities and post-translational modifications (PTMs), in support of the technical development of such non-platform program during cell line selection, formulation selection, and stability studies, etc.

To enable administration of highly potent drug products in the clinic, a closed system transfer device (CSTD) is often used. Recovery and product quality after passing through a CSTD must be evaluated and it can be challenging to measure product attributes at the very low concentrations involved. In this talk, we’ll discuss analytical approaches for assessing concentration and for monitoring product quality for low-concentration in-use samples.

Non-mAb proteins encounter several CMC developability challenges. To expedite these modalities to clinic, CMC and discovery teams collaborate to identify structural weaknesses and degradation pathways of the molecules and enable the selection of the optimal lead. In this presentation, we will share case studies that utilize high-throughput (HT) tools to assess various properties, including the propensity for self-interaction and aggregation, thermal stability, colloidal stability, and biochemical stability.

Single cell characterization is one of the most critical measurements, being utilized in medical diagnostics, cellular therapeutics and biomanufacturing applications. Different cellular therapies based biomanufacturing assays require single cell monitoring and enumeration while determining cellular potency, viability, and activity. Single-cell measurements serve as QC in many biomanufacturing processes. Recently, we developed a 3D printed, autonomous bioanalytical imaging and characterization setup based on florescence microscopy capable of imaging cells at point-of-care.