Category: Formulation and Quality
Purpose: A vaccine drug product, composed by a peptide antigen linked to a protein carrier and mixed with two different adjuvants (Adj.) in appropriate formulation buffer is under study for toxicological and Clinical Trial Manufacturing batches.
Bicinchoninic acid (BCA) assay method was used in early development studies for protein content determination, in order to evaluate the stability of formulated anti-Alzheimer vaccine as it will be prepared and administrated to animal (Tox batch) and human (CTM batch).
The original method was claimed to be suitable for quantitating the protein in the vaccine mixture. Results showed a low % recovery of sample solution in presence of adjuvants. By means of ad hoc designed experiments, it was demonstrated that for the intended purpose, a Complex calibration curve (protein-Adj.1) was necessary in place of the suggested system calibration reference, identified as Bovine Serum Albumin (BSA) in the original method.
Methods: The purpose of the analysis is the determination of the assay by BCA technique, performed using the Thermo Scientific Micro BCA Protein Assay kit. The procedure foresees an incubation step at +37°C for 2 hours and a reading step at 562 nm by means of a Microplate Reader equipped with analysis software.
A calibration curve was built to determine the protein concentration in the mixed vaccine solution, composed as follows: protein/Adj.1/Adj.2 in the ratio of 50:500:562,5. Adj.2 has been removed by centrifugation prior testing.
A protein-only calibration curve was performed in place of BSA curve, in the range from 200 mg/mL to 5mg/mL and each level was analyzed in triplicate. Formulation buffer (10 mM Tris, 250 mM Sucrose, 0.01% Polysorbate 80, pH 8.1) was used as diluting medium, analyzed as blank solution and its contribute has been subtracted to determine the protein concentration. Positive control was prepared at ~100 mg/mL of protein and a mixing sample solution was prepared with ~100 µg/mL of protein, 1000 mg/mL of Adj.1 in NaCl medium (corresponding to Adj.2 matrix).
Protein sample better fits in a protein calibration curve respect to the BSA one but the Adj.1/protein mix shows a high recovery (as with BSA curve). It has been hypothesized an interaction between protein and Adj.1 causing a false positive high concentration.
A new set of experiments were designed by building specific calibration curve: