As potency of a biopharmaceutical product is determined by bioassay relative to a reference standard (RS), the development and establishment of a suitable in-house RS is an important activity. As drug development proceeds, with possible changes in the manufacturing process and formulation, it usually proves necessary to replace an earlier RS with one that is representative of the current production or if stocks are running low. Most drug development programs require the establishment of a succession of RS, starting with a development RS (DRS), followed, as clinical trials start, by an interim RS (IRS) and then, ready for license submission, a primary RS (PRS) and working RS (WRS).

During the early stages of product development it is not possible to foresee all the factors, such as special formulation and storage conditions, that might eventually prove necessary for the preparation of a RS suitable for use at later stages in the program. At early stages of product development, knowledge of the product is limited and, commonly, the bioassay also will be in early development. Additionally, at early stages of product development, resources for development of the RS will be limited, so investigation of the potential RS has to be a compromise between the scientific ideal and the practically feasible. However, the following points should be considered from an early stage, even if they cannot be thoroughly investigated or implemented at that point.

From the outset, it is important to keep retention samples of all RS. Sufficient stock of RS should be held at a second location to permit comparison with a replacement standard as insurance against a catastrophic loss of the stock of the current RS. Periodic comparisons of samples from the two locations should be made to ensure no differences are arising between the two stocks.

The properties required of the RS are not necessarily the same as those for the drug substance or drug product. For some products, it may be possible to use a production batch of vials of the drug product. Sometimes, however, it may prove necessary to select special formulation, processing, container and storage. Although, ideally, the same reference standard should be used for the potency assay and other analytical techniques, different analytical techniques may require different preparations or formulations of the RS.

Formulation and Storage

Stability for long-term storage of the RS may need to be greater than that of the drug product intended for clinical use. In many cases, long-term stability can be enhanced by storage at lower temperatures than that used for the clinical product. For liquid formulations, the RS is commonly stored at -80°C but it must be determined whether the freezing process damages the material. Storage of liquid formulations at -20°C can be risky as this temperature is at the transition between frozen and liquid and freeze-thaw transitions occurring around this temperature may adversely affect stability

For many biologicals, lyophilization can enhance long-term stability. The optimum residual water content and suitable formulation must be determined for each individual case. Too low a residual water content can adversely affect stability. Compositions suitable for liquid formulations may not be suitable for lyophilization and stabilizers may be required. Lyophilization may require investment in infrastructure and investigation of lyophilization and reconstitution processes but can give improved long-term stability and permit storage at higher temperatures (for example, -20°C rather than -80°C). Lyophilized material is not susceptible to freeze-thaw transitions, so this may permit easier handling.

A single bioassay run may require only a small quantity of the drug so it may be desirable to use a small volume or low concentration of the API per aliquot of RS. In this case, stabilizers, carriers or bulking agents may be required. Difference in formulation between the clinical product and RS may cause non-similarity in the dose-response curves. This may be overcome at high dilution or may require compensation in the assay medium.

A variety of stabilizers and bulking agents have been used (for example serum albumin, amino acids, detergents, sugars), but potential formulations will need to be tested for effects on stability and recovery for each individual drug and for interference in the intended bioassay(s).

Container and Closure System

The proposed container and closure system for the RS must be tested to determine its suitability. If it is planned to use the same as for the clinical product, this must be tested for its suitability under the proposed storage conditions. Butyl stoppers, for example, can shrink at low temperatures. Enhancing stability of the RS may be achieved by prevention of gas exchange, moisture entry, reaction with stopper, and reaction or adsorption on the container surface. Suitable containers range from cryovials, stoppered glass vials to heat-sealed glass ampoules, depending on the individual case. The selected aliquot size will influence the container system chosen. For a small volume of reference standard, a large container will have the disadvantages of: a large headspace (increased evaporation, reaction with headspace atmosphere); large surface area (adsorption, reaction with container material); difficulty of recovering standard; costly storage volume requirements.

Filling the headspace with an inert gas, thus reducing the oxygen, may increase stability by reducing oxidative reactions. To be effective, this should be used in combination with a container system that reduces gas exchange.

Protection from light should also be considered and amber or opaque containers may be used.

Size of RS Aliquots

Ideally, each bioassay should use a freshly opened aliquot of the RS. However aliquots may be larger than required for a bioassay: for example, if the RS consists of a batch of vials of the drug product as used in the clinic or if the RS has been prepared in larger aliquots to be suitable for other analytical techniques. If aliquots are larger than required for immediate use, it may be possible to store thawed or reconstituted aliquots for later use, to avoid wastage. Such interim storage may include refreezing liquid formulations, freezing reconstituted lyophilized aliquots, or refrigerated storage, for example 2-4°C. Generally, freeze-thaw cycles should be avoided if possible. If interim storage is contemplated, extensive bioassay and physicochemical analyses must be conducted to determine that the proposed conditions do not impact the suitability of the RS. Any changes detected must be assessed carefully, even if they do not appear to impact the potency in the current bioassay as they could affect the dose-response in different potency assay.

How Many Aliquots to Prepare?

Replacement of a RS is necessary when the current RS is no longer suitable or when stocks become insufficient for the anticipated requirements. The exercise involves significant work and the risk of some degree of discontinuity, so the number of aliquots prepared is a balance between minimising the need for replacement due to shortage of stock and avoiding wastage of valuable product and resources. The number of aliquots required depends on several factors, including the stage of product development. For the DRS and IRS, when the product will probably be undergoing changes in the production process, the RS may need to be replaced so that it remains representative of the current product, while, in contrast, ideally, the PRS will last the lifetime of the commercial product. In estimating how many aliquots of RS will be required, the number required for the product stability testing programs, comparability studies, batch release, as well as those required for stability and comparability studies of the RS itself, plus retention samples, should not be underestimated. On top of anticipated requirements, an overage should be included.

Before Depletion of Stock

It is crucial to avoid running out of the RS at any stage. Keeping track of and initiating a replacement program while there is sufficient remaining stock to allow thorough comparison of the existing and candidate replacement RS can avoid discontinuity and potential interruption of the drug development program. If possible, a candidate replacement RS can be run routinely in the bioassay as a sample, to allow extensive data to be accumulated.

Retention samples of every RS should be held. Comparison of a candidate replacement RS with previous, as well as the current RS, may help prevent drift or identify anomalous characteristics.

It is sometimes possible to prepare a further stock of aliquots of RS from the same batch of product as used previously. However, the handling and storage history of this later set of aliquots will not be identical to that of the first. Rigorous testing is necessary to demonstrate similarity of the initial and later sets of aliquots and aliquots should be clearly identified as to which set they belong.

In conclusion, awareness of various factors that will, or may, need to be investigated later in the RS development program, even if they cannot be addressed initially, can help avoid unpleasant surprises and allow timely preparation for the necessary actions.

For further information:

• The BEBPA Reference Materials Focus Group has been reviewing current practices in the development and establishment of reference standards for potency assays and will be publishing the findings as a white paper on the BEBPA website, bebpa.org.
• BEBPA is holding a virtual conference, 2024 Reference Material Virtual Conference, June 24-28, 2024, with presentations, workshops and round-table discussions. For further information, see the BEBPA website, bebpa.org.