As AAV-based gene therapy is coming of age with clinical success and increasing numbers of market entries, the industry has set out to develop AAV vector production protocols according to CMC standards. We are leveraging our expertise in CMC for therapeutic proteins to adopt existing and integrate new protocols for AAV vector generation, purification, and quality control. A case study will be presented to illustrate the differences between therapeutic modalities.

Distinguishing aggregated API from other particle types is important for understanding the root cause of instability. Existing methods are unreliable, too cumbersome and difficult to use in many workflows. With Aura, you can now finally count, size, and characterize aggregates and identify them as proteins, non-proteins, or other molecules.

Gene therapy products comprise a protein capsid and a nucleic acid packaged inside. We describe how analytical tools traditionally employed by biochemists for protein therapeutics are applied as is, or are adapted to GT products. We highlight similarities between gene therapy and protein-based products and discuss specific examples related to their critical quality attributes. We describe challenges unique to gene therapy products and how they were overcome with new methods.

Immune monitoring provides insight into a patient’s immune response over the course of treatment. The challenge lies in making peptide MHCI complexes, since HLA are highly polymorphic. To successfully produce libraries, highly sensitive assays are necessary to validate peptide exchanges. Here, we describe a robust SEC-MS method and also a sensitive and high-throughput assay using capillary zone electrophoresis coupled to a high-resolution mass spectrometer for characterizing peptide-exchanged MHCIs under native conditions.

The sophistication required for proper molecular design causes the manufacturing and development of bispecific antibodies to be complex. Monitoring structural stability is critical for retention of the favorable drug distribution and pharmacokinetic of the antibody format. Classic ligand binding assays have limited resolution to capture some structural modifications in bispecifics. Here we will discuss characterization of bispecific molecules from pre-dose and post-dose pharmacokinetic samples.

Co-formulated monoclonal antibodies pose new challenges for analytical characterization due to their increased complexity. For example, aggregation is a common degradation pathway for mAbs and is often monitored under native state with size exclusion chromatography (SEC), but for co-formulation, new challenges appear due to similar size and shape of individual mAbs. A 2D-LC approach with orthogonal methods to SEC was adopted to improve product knowledge of single mAb profile in co-formulation and characterize aggregation in co-formulated DPs.

Antibodies are the most rapidly growing form of therapeutic. High-throughput methods of characterization are essential as companies and researchers develop new candidates. Dr. Owen discusses advanced characterization techniques to assess antibody-drug conjugates. For ADCs, the level of payload and site of conjugation are determined using LC/MS. Thermal stability and binding affinity are characterized using DSC, IR, and ITC. These methods can be used for the in-depth analysis of these biologics and for routine product validation.

Antibody-drug conjugates are important classes of molecules currently being used to treat multiple diseases. Advances in small-interfering RNA (siRNA) technology result in numerous RNAi-based therapies being pursued in clinical trials. Herein we present how native nESI-MS, in both positive and negative polarities, is the only bona fide analytical method for accurate intact MW and RAR (RNA-to-antibody ratio) calculation for siRNA-mAb conjugates.

Bispecific conditional dual variable domain immunoglobulins (cDVD-Igs) are targeted and locally activated biologics that offer new prospects for engineering efficacy, while minimizing systemic side effects. We will describe preclinical examples of tissue targeting and activation in in vivo disease models as part of a new generation of locally acting “regio-specific” biologics therapies.

In this talk, we present our work on co-formulation of the model proteins, EPO and G-CSF, which are interesting from both clinical and physicochemical perspectives. These cytokines differ a lot in their structure and respective stability profile, so obtaining a stable co-formulation of both proteins in one solution is challenging. We present a systematic approach to study and stabilize these two physicochemical, very different therapeutic proteins in one formulation.

We will present several case studies following the developability assessment of our preclinical to commercial pipeline. These case studies will illustrate how the evolving field of developability can help inform antibody selection and progression through development. We will highlight several examples of red flags and red herrings in developability and shed light on some of the underlying mechanisms and how they relate to antibody development.