(M1030-12-77) A Study on the Visible Aggregation of a Peptide Formulation Under Stress Conditions at Manufacturing Fill and Finish Process

Purpose: Proteins are large molecules made up of amino acids whose native structure and stability is a result of fine balance between covalent, hydrophobic, electrostatic, hydrogen bonding and van der Waals forces. Change in the protein environment such as change pH, temperature, concentration, shaking/shear and surface interaction encourages partial unfolding of the peptide leading to protein aggregation or physical instability of the formulation. Depending on the kinetic energy of the process, aggregation can be reversible or irreversible leading to soluble or insoluble aggregates. Soluble aggregates refer to those that are not visible as discrete particles and may not be removed by a 0.22 µm filter. Whereas insoluble aggregates may be removed by filtration and are often visible to the unaided eye, as these aggregates can are more likely to elicit an immunological response and possess a greater risk in product stability. Therefore, -this study evaluates the sensitivity of a peptide molecule to temperature and shear generally encountered during manufacturing filtration and fill/finish operations, on visual aggregation.

Methods: Drug product formulation containing an endogenous peptide 0.1 mg/mL and sodium chloride 9 mg/mL in Water for Injection (WFI) was pumped through a filtration/fill tubing (Pt.Cured Silicone) using a peristaltic pump for upto 2hrs. Two rpms 30 and 110 rpm at two different temperatures, RT and 2-8 °C were selected based on the conditions the drug product would be exposed at manufacturing process. After a defined period, the samples were collected and observed visually for formation of visible aggregates along with controls where the drug product was not exposed to any sheer and only held at two different temperature conditions.

Results: Visible aggregates were observed only in the samples that were exposed to a pumping speed of 30 and 110 rpm at RT for 30, 60, 90 and 120 mins, whereas the solutions exposed to shear at 2-8°C appeared clear, for all the experimental conditions studied as presented - in the Table and Figure. The aggregates appeared as white flocculates, that tend to disappear while shaking but would reappear when held still. The control samples placed at RT and 2-8 °C also appeared clear and free of visible particulates indicating that shear and temperature both have an effect of formation of aggregates.

Conclusion: Based on the study it can be concluded that the -studied peptide is sensitive to shear and temperature, which may lead to formation of aggregates and failure of visual inspection. Formation of aggregates can be avoided by filtering and filling the drug product at low temperature and Low shear conditions.

References: Frokjaer, S., Otzen, D. Protein drug stability: a formulation challenge. Nat Rev Drug Discov 4, 298–306 (2005).
Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm. 1999 Aug 20;185(2):129-88.

Table: Visual observation data at different time points

Figure 1: Photographs depicting visual observation of samples exposed to shear conditions at RT, 2-8 °C and control samples.