(T1530-09-56) Points to Consider in the Development of Activity and Potency Assays for Enzyme Biotherapeutics

Purpose: Potency/activity assays play a pivotal role in the development of nearly every biologic and its characterization is expectation of global regulatory agencies. These assays take many forms depending on the mode of action (MOA) of the biologic. One such activity assay is enzymatic activity, which can be applied to recombinant enzymes, in-process enzymes, and gene therapies intended to produce or replace enzymes to directly address a disease. The ability of enzymes to remain active outside their source organism makes them an ideal candidate for biotherapeutics. Some of these therapies include engineered enzymes with different activity profiles than prior therapies or naturally occurring enzymes. The activity of these enzymes can be measured in many ways including absolute activity, specific activity, relative activity, or kinetic activity. The purpose of the current work is to use examples of these forms of enzymes – here in process enzyme and recombinant therapeutic enzyme – to enumerate on challenges and solutions in developing activity assays to monitor the MOA and capability of these proteins to deliver the intended activity. Specifically, designing experiments to optimize determinations, considerations, and approaches on how to execute the assays and the benefits or requirements of comparative assays.

Methods: The enzymes of interest are – a serum protease to treat clot formation in a manner similar to tissue plasminogen activator and an immobilized protease used in the manufacturing process to produce recombinant proteins. Both enzymes are exposed to the substrate in the reaction system. For the serum protease, the activity was measured by optical dispersion and the dissolution of a physical fibrinogen clot. The amplitude and time were key parameters measured for activity determination. Comparisons were made between a drug product and drug substance versus the WHO reference material for tissue plasminogen activator. The drug substance was a free protease, and the drug product was an encapsulated polymeric fluid which converts to gel upon injection. The assay was based on the compendial assay for Alteplase. For the process enzyme the measurement of Trypsin activity was performed by means of substrate cleavage to generate a colored residue called p-nitroaniline (p-NA). The amount of p-NA in nmol per mass was calculated against p-NA reference standard curve generated from standard reagents in a commercially available kit from Millipore Sigma. In both cases kinetic and static data were used to evaluate activity, relative activity, or relative potency as is appropriate.. Calculations were done in Gen5 Secure version 3.02 (Agilent Technologies) and other software capable of more advanced polynomial fits were also evaluated.

Results: The relative and absolute rates of the enzymes were determined for both enzymes under study. Ratios of substrate to protein, durations of data acquisition and normalization methods were explored and optimized. Different normalization means provided different levels of control of the immobilized production enzyme. In addition, the comparison of kinetic versus static (endpoint) determination offered useful insights with different caveats and benefits. The kinetic profile of the manufacturing protease is shown in Figure 1. The Standard curve within the kit is created from cleaved p-NA substrate which makes the comparison of kinetic activity derived from samples to a “static” standard curve erroneous. Therefore, endpoint determination of the Trypsin activity was pursued (Char t1). Evaluations of the serum protease provided the need for creative comparison. The rate constants for each enzyme – WHO, drug substance and drug product – were very different making static and dynamic comparisons challenging. Representative kinetic plots for clot dissolution are shown in Figure 2 for each of the three forms of serum protease. The time dependence demonstrated provided challenges in creating a single assay for evaluation of all three materials. In these instances, normalization to protein for specific activity and static determination provided useful comparative data and interpretive relative activities. Rate constants and kinetic behavior provided great insights into the materials themselves. The use of absolute activity determination proved useful for both enzymes. In both instances assays were made validation ready and were pivotal to the continuation of product development.

Conclusion: Enzyme activity is a critical quality attribute of many biotherapeutics with enzymatic activity as a component of their MOA. Here some of the challenges in dealing with enzymes are discussed and solutions proposed to create quantitative assays for the determination of the relative activity of these materials and we share considerations for the development of the assays and the products. The insights gained from this research contribute to enhancing the rigor and reliability of use for drug products and ancillary materials in biotherapeutic production processes.

Figure 1: Kinetic plot of trypsin activity for immobilized enzyme and trypsin reference material. Trypsin activity derived from the samples appear to plateau within 2 minutes of the reaction.


Figure 2: Kinetic display of clot dissolution by: a) WHO tPA reference; b) protease drug substance; c) protease sustained release drug product