An international collaborative study to replace the 1st international standard for prekallikrein activator

Abstract

Samples B and C were established as the 2nd IS (code 02/168) and PKA activator in albumin BRP batch 1 (Y0000263), respectively, with a potency of 29 IU per ampoule. Results from this study indicate that testing for PKA in albumin may be less sensitive to the source of PKS than previously feared. The study highlights a number of methodological issues that may need revising in the Ph. Eur. general chapter 2.6.15.

Development of an updated assay for prekallikrein activator in albumin and immunoglobulin therapeutics

“…All reactions were carried out in a microtitre plate, and each reaction had a corresponding blank for subtraction on the plate consisting of 10 µl of test solution plus 90 µl of buffer to replace the PKS in the first step. Dose–response curves for standards and tests were performed in duplicate, and results analysed by parallel line bioassay methods as outlined in the Ph Eur and previously [7, 8].…”

“…A widely used assay method for PKA is outlined in the European Pharmacopoeia (Ph Eur) [7]. The level of PKA must be below 35 IU/ml, traceable to the WHO International Standard (IS), which defines the international unit (IU) [8]. Similarly, immunoglobulins are tested for contaminating PKA, with activity traceable back to the same WHO IS.…”

“…It was noted that this rule was not always adhered to, probably to conserve PKS, which is time‐consuming to make in‐house or costly to buy. The study [8] also showed variations in the way participants interpreted blanking in their protocols, which suggested that the general advice given in the Ph Eur method was not entirely clear.…”

“…[9]), outline a relative potency determination against a standard in a two-step procedure: (i) an activation step where PK in prekallikrein substrate (PKS) made from plasma is activated for a fixed time to generate kallikrein; and ii) this solution or a subsample is mixed with chromogenic substrate to estimate kallikrein activity by measuring amidolytic activity. Variations within this basic method were investigated previously in the collaborative study that established the WHO 2nd International Standard (IS) for PKA in albumin [8]. This study identified some deviations from the Ph Eur monograph, for example in the ratio of PKS:sample volume, which is supposed to be 9:1 to avoid artefacts due to different salt concentrations between test samples or tests and standards.…”