Assay Transfers: A Guide to USP <1224>

A bioassay supporting a commercial product often has a lifespan of several years or even decades. Over that time, it is often necessary that the assay be performed in a number of different laboratories for a variety of reasons. And, in each, the same rigorous standards must be held to ensure that the safety and efficacy of the product is maintained whether the assay is performed in Sydney or San Francisco. When an assay is moved from one site to another, it is important that an assay transfer procedure is followed. Known formally as a Transfer of Analytical Procedure or TAP, these are governed by USP Chapter <1224>.

Once validated, it is advisable for the lab(s) in which an assay is performed to remain consistent. This is to minimise variance in the assay results: different labs will inevitably have different environmental factors and operators which can introduce variability into measurements. This is not always possible, however, and it is common that an assay need be moved once or indeed several times over its lifespan. There are several reasons why this may be necessary:

• The assay is moved to a higher capacity laboratory

Manufacturing requirements fluctuate regularly, influenced by the forces of supply and demand. There might, therefore, arise a scenario where the testing capability of the lab at which an assay was originally validated is not high enough to meet increased manufacturing production. One solution might be to move the assay to a lab with higher capacity, which would require a transfer study.

• Business concerns require a change of laboratory

On occasion, it might be deemed necessary to move an assay to a site with an improved supply chain for key reagents or access to the labour market. Or an external factor may force an assay to be moved, such as a technical failure or natural disaster at the original facility which prevents the running of the assay.

• Mergers and Acquisitions

Mergers and acquisitions are very common in biopharma, with the potential for a bioassay changing hands several times over its lifespan. While the facility which runs an assay can also be acquired in such a deal, it might be necessary to move the assay to a new facility once deal is complete.

Changing the facility in which an assay is run has the potential to introduce problems. The process of validating an assay formally establishes its expected accuracy and precision, as well as formalising limits on suitability and acceptance criteria for the assay going forward.In its most basic form, validation procedures only establish these key properties for a single facility. When we move the assay, therefore, we cannot be sure that the assay will continue to perform as expected. This can be as simple as ensuring that operators at the new facility fully understand the assay procedure and that all steps in the assay SOP are well described. A misunderstanding about something as basic as incubator temperature, for example, could be the difference between a well-behaved assay and completely unusable results. Changes in the equipment used for the assay might also present challenges. If the assay is to be moved from a human-operated lab to an automation lab, for example, certain suitability tests might be influenced by the increase in precision.

At their heart, therefore, TAPs are required to ensure that the quality of the assay which was established at its original facility is maintained at the new lab. This can be in relation to procedural concerns, as we have described, but we also need to make sure the results are comparable between the RU and the SU. This is essential for ensuring the product continues to be safe and efficacious after the transfer.

There are three main types of assay transfer outlined in USP <1224>. These are:

Comparative testing

Lots of product are tested both at the old lab (the sending unit, or SU) and at the new lab (receiving unit, or RU) and the results are compared.

Co-validation

The validation process for the assay involves both the SU and the RU. This means that the RU will automatically be qualified to run the assay . Clearly, this can only take place if the assay transfer is planned or anticipated ahead of validation of the assay.

Complete/partial validation of the assay at the RU

Complete or partial validation of the assay are alternatives to a comparative study. Partial validation is uncommon, but can be justified in a limited set of circumstances. For example, one might be performed at the RU when transferring the assay involves minor changes to the assay method which do not require a full re-validation, and a comparative study is deemed inappropriate. The extent of the partial validation will depend on the nature of the modifications. A more detailed description of co-validation and partial validation procedures is given in USP <1225>.

In some rare circumstances, a TAP can be avoided entirely using a transfer waiver. In such cases, the RU is considered to be qualified to perform the assay procedure without any further testing. Some scenarios in which a transfer waiver may be appropriate are:

• The new product is comparable to an already existing product, and is analysed using procedures with which the RU already has experience
• The procedure for the transferred assay is described in regulatory guidance and is unchanged
• The transferred assay is very similar or identical to one already in use
• The personnel responsible for running the assay at the SU move with the assay to the RU

If the assay is eligible for a transfer waiver, then this should be documented by the RU with appropriate justification.

More commonly, an assay transfer will require comparative testing at the new lab. Designing such a study has many elements that need to be considered from a statistical perspective in order to ensure that it has a reasonable chance of success. Failure to address these in advance may result in unnecessary study failure.

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There are two main aims of a TAP using comparative testing. The first is to establish that the results obtained at the RU are comparable to those from the SU. Second, it must be determined that the precision of those results is acceptable.

In such cases, one can use repeated measurements of a single lot of product at both the SU and the RU to establish the comparability and precision of the results. This is because a TAP is not intended to evaluate the full manufacturing procedure, just whether the results obtained by the RU are similar enough to those from the SU to be acceptable. In many cases, however, several lots with a range of potencies are tested to ensure that the comparability and precision extends over the full range of the assay.

Comparability

We test the comparability between the results from the SU and RU to establish that the measurements are sufficiently close to each other. This is usually done by making repeated relative potency measurements of the same sample at the SU and the RU. The geometric mean of these measurements for each unit are found, and compared by taking a ratio. The closer the ratio is to one, the more comparable the results.

Of course, we need to formalise how comparable the results must be to be considered acceptable by defining acceptance limits. These will depend on the nature of the product, the specification limits, and the variability of the assay. If the 90% CI of the ratio falls outside of the chosen limits, we do not have evidence that the results are comparable between the SU and the RU, even if that CI contains one.

Variability

Unlike the comparability, only the RU is relevant when establishing acceptable variability in an assay transfer. This is because the variability of the assay at the SU will have already been determined to be acceptable either during the original validation of the assay or a previous transfer. Our goal is, therefore, to look at the results we obtain from measurements at the RU only and find whether their variability is acceptable.

The variability of the assay at the RU is measured using the Intermediate Precision (IP) of a series of results taken at the RU. The IP encapsulates the variability one would expect from several repeated runs of the assay. That is, both the within-assay and between-assay variability is included. The USP guidance suggests that the Percentage Relative Standard Deviation (%RSD) of the measure relative potencies is used to report the IP. This is essentially the same as the Coefficient of Variation (CV). For data such as relative potency, however,  the Geometric Coefficient of Variation (GCV)is appropriate as it is log-normally distributed.

In order to be acceptable, the measure of intermediate precision must fall below a specified upper limit. This can either be placed on the value of the intermediate precision, or, ideally, on the upper limit of a confidence interval found on the intermediate precision. For example, we might set an upper limit of 12%, below which the upper 90% CI limit must fall for the precision to be considered acceptable. Unlike the comparability of the measurements, we only put an upper bound on the intermediate precision: we always want the measurements to be as precise as possible.

Finding the appropriate sample size for a comparative study is a complicated process, and often depends very strongly on the exact circumstances of the study. We always want to design a transfer study with a high probability of succeeding. In general, the required number of runs in the study will depend on the expected comparability and precision of the results. A highly variable assay will often require far more runs than a less variable assay to achieve the same probability of success.

It is important to make a determination of how many runs might be required for a comparative study early in the process of planning an assay transfer. In some cases, the number of runs will be high enough that a partial or even full re-validation would be less work for the RU.

Alongside the statistical study, consideration should be made to ensuring that the documentation surrounding the assay is sufficiently detailed. This allows the transition from the SU to the RU to be as smooth as possible.

The USP guidance outlines that the assay procedure should have written instructions detailed enough that “a trained analyst should be able to perform it without difficulty”. It is also recommended that a meeting between the teams responsible for the assay at the SU and RU be held pre-transfer to identify any areas which require clarification. Indeed, it is suggested that having a member of the team who developed and/or validated the assay on-site during the transfer is often beneficial.

In some cases, the requirement for an assay transfer might be unexpected, particularly in the case of a merger or acquisition. In many, however, the transfer will be anticipated long in advance, particularly when an assay is being developed at one facility before being sent to another to be used to support manufacturing. While statistical involvement in both these scenarios is vital, early involvement in the latter can prove hugely beneficial. This can help ensure that the transfer forms an integrated part of the lifecycle of the assay, rather than a hurdle which needs to be jumped.