Revision of USP Chapter <1033> on the Validation of Bioassays

In the news "Revision of USP Chapter <1033> on Validation of Biological Assays" we had already reported on the second attempt to revise USP chapter <1033> on the validation of biological assays, which made the first approach from 2022 obsolete. In summer 2026, a final version was published without further consultation and will become official on 1 December 2026. The chapter complements the USP chapter series on bioassays, which also includes <1032> and <1034>, about which we reported in recent newsletters.

Objective and Background

The revised chapter describes how biological assays used to determine relative potency, or relative biological activity, against a reference standard should be validated so that their suitability for the intended use - in particular for reliable quality and release decisions - is demonstrably documented. It supplements the general validation principles for physicochemical methods with the specific features of biological potency measurements.

The background is that these assays rely on biological substrates or reagents and can therefore be inherently more variable than classical chemical analyses. The scope is not limited to tests with living cells or animals; it also includes enzymatic activity and binding assays, provided they determine relative potency on the basis of several concentrations or dilutions of test material and reference standard.

Validation is understood as part of a lifecycle: method development and definition of the SOP are followed by validation, and ongoing performance monitoring maintains suitability in routine operation. Fitness for purpose is decisive: validation requirements should be based on whether the specific procedure delivers reliable results with sufficient accuracy and precision for the intended decision. To this end, the chapter distinguishes between the method, which generates a measurement, and the procedure design, which turns this into a reportable result - including the replication strategy.

Summary of Contents

The chapter provides recommendations on planning and documenting the validation, on selecting samples and validation runs, and on considering relevant influencing factors. The focus is on relative accuracy and precision; it also covers dilution linearity, the suitability of the dose-response model, the consistency of the potency estimate across the working range, and specificity and selectivity. To demonstrate fitness for purpose, relative accuracy and precision are considered more closely together in terms of the Total Analytical Error (TAE). Risk-based statistical approaches - in particular prediction and tolerance intervals, and a study size aligned with them - help to assess the risk of wrong decisions in quality and release decisions and to demonstrate the suitability of the procedure in a traceable way.

The chapter can be accessed on the USP website after free registration, which is subject to a fee. For those wishing to explore the topic of <1033> as wall as the chapters <1032> and >1034>  in greater depth, PharmaLab 2026 offers a two-day track on bioassays and potency assays, providing an opportunity to discuss the new chapters with representatives from the USP and other European regulatory authorities.