CHARACTERIZATION CORNER - A New Weapon in Formulation Development

By: Matt McGann, Field Applications & Marketing Manager, RedShift BioAnalytics, Inc.

At the 2019 Colorado Conference on Protein Stability held in Breckenridge CO, industry experts, key opinion leaders, and researchers gathered to discuss challenges and advances in formulation development of biologics. Among the work presented, several studies using computational modeling to elucidate formulation stability indicated that our understanding is still a long way from being prescriptive in formulation design. When discussing aggregation and amyloidosis, it was highlighted that the role of reversible self-association and aggregation is far more complex than envisioned. Many talks highlighted the breadth of analytical technologies that are being used to understand the complexities of biopharmaceutical design and development. A significant observation throughout the conference was that sometimes the most stable formulation is not, in fact, the most suitable, as it is ultimately more important to formulate a drug product that is fit for purpose.¹ A good example of this is the use of citrate buffer, which is known to cause discomfort at the injection site in patients.²

Formulation development scientists have a core set of technologies used by almost every researcher; there is however no standard set of protocols used industry wide.³ It is also evident there are several analytical gaps within the toolset, particularly in relation to high-concentration formulation development, where nearly all analytical techniques currently fall short.

Critical factors in the design of a biological drug substance include dosage form, concentration, route of administration, type of API, and type of formulation.¹ Additional factors related to manufacturing process, fill-finish capabilities, and container closure system will also provide limitations on what can and cannot be utilized in a formulation. The types of excipients that are typically used for stability, solubility, and pH control purposes include buffers, salts, sugars, surfactants, and amino acids. Additionally, excipients such as antioxidants and preservatives can be added to ensure the shelf-life of the product.¹

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When assessing the suitability of a formulation, there are generally three aspects that need to be considered:

1) Colloidal stability, a measure of the interactions between proteins in solution, and between proteins and the solvent. These interactions can be based on hydrogen bonding, Van der Waals interactions, and dipole-dipole interactions. Repulsive interactions between particles, measured by a positive diffusion interaction parameter (kD) and second virial coefficient (B22) are generally considered to be two primary indications of such stability. Additionally, physical charge measurements, such as effective charge (Zeff) or isoelectric point (pI), can be used to assess colloidal stability.

2) Chemical stability, a measure of the resistance of the protein to chemical denaturation processes, such as oxidation, deamidation, and disulphide cross-linking. The development of charge variants of a protein as a result of any of these processes can be observed using techniques such as capillary isoelectric focusing. Additionally, aggregation temperature and the molecular weight envelope of a protein can highlight changes in chemical structure. Most of these techniques can be measured in a high-throughput or at least in a fully automated fashion, making them suitable for screening larger numbers of variations in a formulation set.

3) Measurement of conformational stability of formulated proteins by assessing melting temperature, or higher order structure poses a challenge in terms of both throughput and compatibility. Whilst DSF offers significant throughput, it only provides a global measure of the conformational stability of the structure.⁴ Direct measurement of higher order structure using far-UV CD and FTIR provide information regarding the secondary structure of a protein; however, both have limitations. Circular dichroism works most effectively at concentrations less than 2 mg/mL and is not compatible with many excipients and buffers. FTIR, on the other hand, offers better chemical compatibility and concentration range; however, volume limitations and a lack of automation make it a challenge to implement.

With the requirements of formulation development of biopharmaceuticals becoming more demanding, evolution of analytical tools that can measure stability over a wide concentration range, in the presence of complex buffer systems is critical. Microfluidic Modulation Spectroscopy is an alternative technique that combines microfluidics with a high-power mid-infrared laser to quantify secondary structure across a concentration range of 0.1 to over 200 mg/mL in complex formulations. The technology provides higher order structure information in complex formulations without the need for dilution, buffer exchange, or chemical modification, and is the final addition in the biophysical characterization scientist’s toolkit.

REFERENCES 

1. Weinbuch, D., Hawe, A., Jiskoot, W. & Friess, W. Introduction into Formulation Development of Biologics. in 3–22 (2018). doi:10.1007/978-3-319-90603-4_1.
2. Laursen, T., Hansen, B. & Fisker, S. Pain perception after subcutaneous injections of media containing different buffers. Basic Clin. Pharmacol. Toxicol. 98, 218–221 (2006).
3. Smith, M. K., French, J. L., Kowalski, K. G., Hutmacher, M. M. & Ewy, W. Quality by Design for Biopharmaceutical Drug Product Development. in Springer 18, 710 (2015).
4. Parr, M. K., Montacir, O. & Montacir, H. Physicochemical characterization of biopharmaceuticals. J. Pharm. Biomed. Anal. 130, 366–389 (2016).

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