To ensure a long product shelf-life for biopharmaceutical drug products, a stable storage environment is critical. Determination of the optimal environment is initiated early in the development process through preformulation screening. In order to maintain potency and activity of protein active pharmaceutical ingredients (APIs), preservation of structural conformation—a characteristic highly influenced by formulation environment—is essential. However, analytical monitoring of this parameter during preformulation screening is frequently omitted. In this article we describe the use of various biophysical techniques as formulation screening tools, aiding elucidation of protein conformation and protein dynamics. We will also demonstrate the importance of incorporating these techniques in early-stage protein screening studies.

During preformulation screening, even small changes in buffer or excipient content and composition can produce changes in conformation that are initially slight, but over time can result in significant alterations in molecular structure. Small changes in protein folding can result in exposure of hydrophobic regions, triggering a cascade of degradation events such as deamidation, oxidation, and aggregation that gain momentum over time. Therefore, even small structural changes should be investigated and corrected through formulation design early in the drug development process so as to minimise potential problems at later stages.

Frequently used techniques for monitoring the conformational stability of a protein drug substance and drug product throughout manufacturing and storage include preformulation screening studies such as characterisation and comparability studies. Protein conformation analyses are rarely conducted, likely due to the inherent insensitivity of the techniques commonly employed. Methods such as far-UV and near-UV circular dichroism (CD) are often used to provide secondary and tertiary structure profiles, but these are not typically indicative of stability. New techniques have emerged—some more established than others—that are much more sensitive to small structural changes compared with CD-based techniques. These techniques, including intrinsic and extrinsic fluorescence and differential scanning calorimetry (DSC), greatly improve the effectiveness of early-stage screening studies.

Higher Order Structure Analytical Monitoring During Lead Candidate Screening

As preformulation screening progresses, lead candidate formulations emerge and the complexity of the analytical techniques used to monitor protein stability increase, yielding more detailed information about protein structure and degradation events. During this lead candidate screening process, analyses of secondary and tertiary structure can be conducted. It is important to note that, although secondary and tertiary structural analyses provide assurance that overall structural conformation is maintained, these analyses are often unable to detect minor changes, thus making determination of the optimal formulation difficult. Figures 1 and 2 depict the profiles obtained from far-UV (Figure 1) and near-UV (Figure 2) CD analysis of IgG formulated in phosphate-buffered saline (PBS) and Tris buffer, respectively. Overlaying the profiles demonstrates that the profiles are very similar, indicating that no differences in conformation of secondary and tertiary structures exists between IgG formulated in PBS versus Tris buffer.



Second derivative analysis, which is shown in Figure 3 below, also yields profiles that, when overlaid, are nearly identical. Similar to far-and near-UV DC, the results of second derivative analysis also indicate that the structural integrity of IgG is similar when stored in PBS vs. being stored in Tris buffer.


These data collectively support the idea that the formulation buffers provide an equally stable environment for the protein. However, analyzing the same samples with Fourier transform-infra red spectroscopy (FT-IR), differential scanning calorimetry (DSC), or intrinsic and extrinsic ANS (8-anilino-1-naphthalenesulfonic acid) fluorescence indicates that small, but detectable, changes exist between the two IgG samples.

Figure 4 shows that the overlaid spectroscopic profiles of FT-IR analysis of the same IgG samples are slightly different, indicating that this method is sensitive enough to detect very subtle changes in secondary structure. The increased sensitivity likely results from the bias in the mathematical algorithm employed with beta-sheets, which are major components of IgG secondary structure. Intrinsic ANS fluorescence profiles also exhibited differences of > 3 nm between the wavelength of maximum ANS fluorescence emission between the two formulations (Figure 5), demonstrating the ability of this technique to detect more subtle conformational shifts. The blue-shifted ANS fluorescence spectrum of IgG in PBS suggests that the antibody in this formulation contains a greater number of accessible hydrophobic regions than that in the Tris buffer, indicating a reduction in conformational stability of the protein formulated in PBS compared to Tris.  This observation is confirmed when the same samples are analyzed by DSC (Figure 6), for which the PBS-formulated protein exhibited a lower melting temperature compared to the Tris-formulated protein.




The Importance of Selecting Stability-Indicating Higher Order Analytical Methods during Formulation Studies

Collectively, these data indicate that, although a portfolio of higher structure techniques available for analysis of protein APIs exists, not all of these are stability indicating, which is a key element required for formulation screening. Choosing methods that yield more detailed results is essential for identifying subtle differences in conformational structure, and thus is critical to determine the optimal formulation of a drug. This parameter is frequently excluded from formulation screening designs due to the lack of sensitivity of more well known higher order structure techniques and possibly due to a lack of understanding of the more sensitive techniques that have emerged.

It is important to consider that even superficial changes at initial timepoints can be exacerbated over time and become significant degradation events. Unfortunately, these degradation events sometimes remain undetected until late in the drug development program, after significantly time-consuming and costly procedures have been completed. Late-stage identification of formulation issues is one of the most expensive—and often preventable—eventualities encountered in protein API drug development programs. These issues significantly complicate and potentially jeopardize time-intensive procedures such as reference standard generation and characterisation, as well as stability studies, method validation, and others.

SGS M-Scan now provides a full formulation screening service that includes formulation study design, as well as sample preparation, storage, and analysis incorporating a wide range of stability-indicating techniques. These services enable identification of the optimal formulation environment for both early- and late-stage protein biopharmaceutical products for a range of routes of administration (e.g. topical, subcutaneous or intravenous, etc.)


Dr. Tara Sanderson
Formulation Services Manager
SGS M-Scan Ltd


ICH Q5C. Quality of Biotechnological Products: Stability Testing of  Biotechnological/Biological Products

Caves MS, Barnard MC, Rodriguez-Mendieta I, Millichip MI.  An Orthogonal Approach To Formulation And Stability Studies. Poster presented at: MIBio; 2012 October 30,; Cambridge, UK