The client requested that we develop a vial-lyophilized product for toxicology and clinical safety studies. The product development
time-line was short — we had only six months from the delivery of the first samples of bulk drug substance to the development
laboratory. The client asked that we provide them with a final formulation, a manufacturing process, and validated product
The protein's properties were virtually unknown. At project initiation, the client could not provide solubility data, stability
data, or the protein's isoelectric point. The API contract manufacturer did supply conditions for an RPC method. However,
the reverse-phase method did not resolve major degradation products from the main peak and was, therefore, not useful as a
stability-indicating assay. Analytical method development was required not only for support of early formulation development,
but also for early stability studies, and, of course, for final release assays. Analytical method development occurred concurrently
with formulation development.
Preliminary Reference Standard
Because the analytical methods were evolving as rapidly as the formulation, a preliminary reference standard was established
and analyzed with formulation samples. The preliminary reference standard was prepared by freezing (-70∞C) a solution of the
API in small aliquots. The manufacturer provided the concentration of the API. Because an extinction coefficient was not provided
for UV-Vis concentration determination, all concentration estimations were based on HPLC analysis using the preliminary reference
standard. When we received an extinction coefficient from the client, UV-Vis was used for concentration determination.
The first step we took in analytical support of the formulation development was to determine the protein's isoelectric point
(pI) by gel IEF. The pI is the solution pH at which a protein carries no net charge. In solutions with moderate salt concentration,
the solubility of most proteins is, at a minimum, near their pI.3 An electric field is applied to a gel matrix containing a pH gradient to separate proteins on the basis of their charge.
IEF can be one of the most powerful separation methods for evaluating charge differences and can resolve a single charge difference
between large protein molecules.4