Monoclonal antibodies and novel bispecific DART® molecules are being developed for a variety of indications including immune-oncology. Stage-appropriate and risk-based analytical control strategy needs to be developed to ensure product quality as molecules progress from early to late stages of development. This presentation will discuss analytical method development and high throughput characterization approaches during early stage of development using the above molecules as case studies.

Accelerating early development of biotherapeutics to enable a fast timeline to first-in-human trials has been of interest across the industry. This presentation discusses the analytical strategies and opportunities to accelerate program development timelines while maintaining product quality, safety, and efficacy. These include establishing efficient characterization plan via prior knowledge, utilizing high-throughput assays and automation, and developing platform analytical methods and molecule-specific methods to meet product quality requirements. 

A preclinical candidate was selected from a diverse pool of engineered protease constructs expressed in mammalian and bacterial hosts. Selection from this diverse pool required generic analytical methods and candidate ranking based on developability/QbD principles with an emphasis on production levels and intrinsic stability. Analytical methods were further developed as the program progressed with a fit-for-purpose approach based on performance monitoring and streamlined method verification.

Biotherapeutic developability assessment (DA) represent a new industry trend. At Denali, we apply a series of state-of-the-art analytical and biophysical technologies to carry out these molecular assessments as early as possible in the preclinical stage of development to select the candidate with the most stable profile. Here we present a case study where we use these techniques to characterize the biophysical stability of therapeutic proteins.

Since the introduction of the first recombinant DNA-derived insulin in the 1980s and the launch of Interferons and Interleukins in the 1990s, the biotherapeutics market has shown steady growth. Given significant challenges in the treatment of many life-threatening diseases, biotherapeutics are becoming increasingly complex. Integrated analytical strategies are required to address potential issues in developability assessment for such complex molecules. This presentation will discuss recent developments in leveraging protein analytics for elucidating key aspects of molecular developability, including post-translational modifications and higher-order structure using examples encountered in bispecific antibodies and fusion proteins.

Early developability assessment of biopharmaceutical drug candidates, using rational methodologies and computational methods, can assist in reducing stability risks during development in a cost-effective way.  In this presentation, the author will discuss one potential algorithm to illustrate how developability strategies can be introduced in practical terms during early protein drug development in order to mitigate risks, and ultimately increase the robustness of the biopharmaceutical.

“First in human” biologic programs often rely upon rapid development of GMP suitable process, analytics and formulation, to reach Phase 1 trials as quickly as possible.  Complex programs, (co-formulated therapeutics, multi-valent vaccines) can require parallel development of multiple components.  This talk discusses potential risks of parallelism in early stage development, specifically formulation, analytical and stability challenges that arise and strategies to overcome them.

Buffer exchange is a critical step during formulation development, involving exchange of buffers and concentration of protein solutions utilizing a membrane-based system. However, the process is labor-intensive and time-consuming, requiring multiple cycles of manual buffer refills and mixing. In this study, an automated buffer exchange workflow in a 96 well format is implemented using Big Tuna and evaluated using a model protein. The efficiency of the buffer exchange process is evaluated by such parameters as volume, osmolality, and pH of the prepared protein solution. The quality attributes of the exchanged protein solution are examined by multiple biophysical and analytical approaches, e.g., DSC, DSF, SEC-HPLC, and DLS. Moreover, the samples prepared by this process are compared to those made by standard dialysis process in terms of quality, materials requirement, and efficiency. Implementation of automation enables early-stage, high-throughput formulation screening in a timely manner. Application of automation in early formulation development in conjunction with DOE could become a valuable alternative to platform formulations, especially for more challenging protein scaffolds.

Determining the development risk of an IgG1 mAb therapeutic is relatively straightforward. However, multi-specific antibodies, Fc-fusion, Fab, or other novel modalities can present many analytical challenges. As more complex modalities enter the biologics drug development landscape, developability assessments must remain flexible to address the challenges that they exhibit. This talk will highlight several case studies where biophysical characterization has been instrumental in determining risk.

The presentation will focus on major challenges in formulation development, analytical method development and stability assessment of biologics drug candidates in early phase clinical trials.  Mitigation strategies used to stabilize the biologics will be discussed.  Close collaboration of formulation scientists and analytical scientists are essential to ensure quality and successful product development within aggressive timelines.  Case studies will be included in the presentation.

In biologics formulation development, particle formation in protein solutions is often monitored over 2-3 years. During long-term storage under refrigerated conditions, therapeutic proteins in liquid formulations can form particles after a lag-time ranging from days to over a year. This type of particle formation is often of the nature of crystalline aggregation and not predicted by protein structural stability assays. Early assessment of the risk of particle formation during storage is highly desirable for developability evaluation and pre-formulation development. Based on the fundamental relationship between protein crystallization and liquid-liquid phase separation, we have developed a method to predict the thermodynamic condition for crystalline particle formation in monoclonal antibody solutions. This method provides an efficient means to quickly evaluate protein solubility with low sample assumption and is especially useful for the early development of concentrated therapeutic protein formulations.