Determining Potency of Preclinical Dose Formulations

Potency is a required measurement to determine the amount of active ingredient contained in a preclinical dose formulation. Assessing potency ensures that the test system receives the appropriate amount of active ingredient based on predetermined specifications. Potency determinations are made using a validated analytical method.

Preclinical dose formulation potency

Assessing the potency of preclinical dose formulation is completed by sampling the prepared formulation and assaying using a validated analytical method. Each dosing concentration is sampled and assayed; typically, assays are completed in duplicate. The observed concentration is compared to the theoretical amount and a percent of the theoretical concentration is determined. Typical acceptance criteria are listed in Table I.

Table I: Typical acceptance criteria for different formulation types.

In the event that a dose formulation does not meet the predetermined acceptance criteria, the result must be investigated for laboratory error. If an analytical error cannot be discovered, the effect on the study must be determined.

Each dosage concentration, including control samples, should be assessed for the first and last test batches of in vivo studies, at a minimum. Theoretical concentrations considering displacement factor and density will aid in achieving the targeted concentration, but measuring the actual result of a formulation will detect the true potency level of the drug in vehicle. Conversely, achieving the correct potency level is not always a simple addition of active ingredient to vehicle. The use of laboratory equipment, filtration, compound characteristics, storage, and chemical instability, including weighing and mixing procedures, are factors that can affect potency.

Mixing

Proper and appropriate mixing of a compound is essential to ensure adequate potency and homogeneity of the ingredient in the formulation. However, assumptions regarding solubility frequently exist when preparing a simple formulation. For example, a formulation prepared as a solution may appear soluble; however, results can dictate otherwise. Such an occurrence was observed in a high-range quality control sample preparation shown in Table II.

Table II: Low recovery observed in a high-range quality control sample preparation.

A laboratory investigation was conducted to identify an assignable cause for the low recoveries. A secondary dilution was prepared from the primary dilution as the method instructed. This time, however, recoveries were within specification of 100% ±10. Although the solution appeared to be a true solution, it was clear that the formulation presented problematic mixing and/or dissolution. Furthermore, in a consecutive run, precipitate was later observed in the primary dilution, indicating the potential problem was dissolution of the analyte in the primary dilution. The analytical method was updated to include in the processing procedure that adequate mixing must be performed after the primary dilution to assure complete dissolution, because particles of the analyte may be present. Thereafter, all samples passed the solution criterion. In this instance, the formulation itself achieved the targeted potency; the problem arose during sample processing for analysis. Though the test system did receive the correct dosage potency, it is necessary to have the analytical data to support this conclusion.

Equally important when carrying out many mixing procedures, especially sonication, is allowing the formulation to cool before performing any additional aliquots. Neglecting this in itself can cause low recoveries when diluting. Special mixing considerations are also necessary when working with analytes that are not small molecules. Cautious inversion can effectively mix large molecules and proteins, without potential destructive effects observed from vigorous mixing procedures.

Laboratory equipment

Another factor to consider in achieving accurate potency is the effect of laboratory glassware and/or equipment. Interactions may occur between the analyte and surface of the volumetric flask used. Such an observation was discovered in preparation of samples in plastic versus glass (see Table III).

Table III: Recovery when sample was prepared in plastic versus glass.

Solutions were left in plastic for 30 minutes. As demonstrated, the analyte possessed a high affinity for plastic. Furthermore, an assessment made using a glass serological pipette yielded higher recoveries, as compared with using a positive displacement pipette containing a plastic tip.

Filtration

Filtration is an effective method for removing impurities from a solution; however, if the correct pore size and/or media are not used, the analyte may be removed along with the impurities. An experiment was conducted on a clear, colorless solution. The solution was filtered through a 0.22 µm polyvinylidene fluoride (PVDF) syringe filter, and analyzed prefiltration and postfiltration. At low and high levels, the prefiltered solution had an average percent recovery of 97.1% and 97.7%, respectively. Postfiltered samples had 0% recovery at both low and high concentrations because the analyte was removed by the filter.

Compound characteristics

If potency issues exist after assessing most potential mixing complications, it is important to refer back to the compound characteristics. If a compound is micronized, problematic weighing may exist because of the static, cohesiveness, and/or lightness of the material. Alternatively, the material may be highly hygroscopic and require the use of a desiccant in storage. When weighing a hygroscopic material, it is essential to consider the amount of water being absorbed, as this can cause uncertainty during the weighing process. It is also significant to account for a correction factor in consideration of salt factors and purity. This is important when considering manufactured lots used for in vivo studies that do not undergo purification processes that are performed for clinical trials.

Storage

A final factor in achieving correct potency is a consideration of storage and stability of the formulated compound. It is necessary to have data to support conditions and duration of storage. Degradation of a compound can be seen at different temperature conditions (e.g., ambient, refrigerated, frozen or ultra-frozen). Yellow light versus ambient light may also affect potency if the formulated test article requires protection from light. Analysis of potency of a formulated drug in specified storage conditions, extending longer than the dose formulation, should be performed before dosing. Covering the time window from preparation to dosing ensures that the proper potency of drug is delivered to the test system.

Conclusion

Potency of a dose formulation is an essential and crucial component in ensuring that the test system receives the appropriate amount of active ingredient based on predetermined specifications. If the correct potency is not achieved, the toxicological effects of the drug may not be observed. Failure to achieve accurate potency levels may be affected by many factors including weighing and mixing procedures, use of laboratory equipment, filtration, and even compound characteristics and storage. When these problems occur, it is important to isolate and investigate each variable to identify the root cause.

Ashley Sanchez is an ssociate scientist, Melissa Whitsel is analytical manager, and Amy Smith is director of Analytical Laboratory Operations, all at MPI Research, Mattawan, MI.

References

Code of Federal Regulations Title 21, Food and Drugs (Govenment Printing Office, Washington DC), Part 58.

M. Whitmire et al., The AAPS Journal, 12 (4), 628–634 (2010).