High-Throughput Biophysical Analysis of Protein Therapeutics to Examine Interrelationships Between Aggregate Formation and Conformational Stability

Abstract

Stabilization and formulation of therapeutic proteins against physical instability, both structural alterations and aggregation, is particularly challenging not only due to each protein's unique physicochemical characteristics but also their susceptibility to the surrounding milieu (pH, ionic strength, excipients, etc.) as well as various environmental stresses (temperature, agitation, lyophilization, etc.). The use of high-throughput techniques can significantly aid in the evaluation of stabilizing solution conditions by permitting a more rapid evaluation of a large matrix of possible combinations. In this mini-review, we discuss both key physical degradation pathways observed for protein-based drugs and the utility of various high-throughput biophysical techniques to aid in protein formulation development to minimize their occurrence. We then focus on four illustrative case studies with therapeutic protein candidates of varying sizes, shapes and physicochemical properties to explore different analytical challenges in monitoring protein physical instability. These include an IgG2 monoclonal antibody, an albumin-fusion protein, a recombinant pentameric plasma glycoprotein, and an antibody fragment (Fab). Future challenges and opportunities to improve and apply high-throughput approaches to protein formulation development are also discussed.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

“…To address these questions, we first characterized the physical stability of wild-type and biotinylated GroEL proteins as a function of pH (3)(4)(5)(6)(7)(8) and temperature (10-87.5 C) with a variety of biophysical techniques (Fig. 1).…”

“…Within the discovery phase of pharmaceutical research, this goal is crucial during the testing of a new drug's ability to interact with protein based targets and as part of the design of therapeutic strategies to correct aberrant protein folding encountered in human disease. 3,4 During pharmaceutical development, ensuring the structural integrity of therapeutic proteins during their manufacturing scale-up, long-term storage, and administration to patients is a critical goal of formulation development [5][6][7] and comparability assessments. 8,9 Protein instability issues can affect the success of a protein drug's clinical development due to insolubility, aggregate formation, and chemical degradation along with concomitant loss of potency and increased potential for enhanced immune reactions.…”

Biophysical characterization of a model antibody drug conjugate

“…When antibodies and drugs are combined to make antibody-drug conjugates (ADC), also known as armed antibodies, the conjugation of what is often a hydrophobic drug to the antibody has the potential to alter the chemical and physical properties of the ADC. Pharmaceutical proteins, including antibodies, require extensive characterization of their structural properties, such as aggregation, conformation and stability (9)(10)(11)(12). Such characterization is also required for ADC.…”