How to Assess Preclinical Dose Formulation Homogeneity

Homogeneity or uniformity of a dose formulation is a requirement for ensuring integrity of regulated preclinical safety studies. Demonstrating homogeneity ensures that the preclinical dose formulation contains active ingredient(s) uniformly throughout the formulation, thereby ensuring that the test system is being administered using the appropriate amount of test material. Accurate assessment of dose formulation homogeneity is accomplished by using an appropriate sampling protocol and validated analytical methodology.

Assessment

Assessing homogeneity for preclinical dose formulations is generally completed by sampling the prepared preclinical dose formulation at various strata in the formulation container. Sampling from the top, middle, and bottom followed by analysis using a validated analytical method is an example. Homogeneity is established by sampling from the initial preparation at both the lowest and highest dosing concentrations. Confirmation of homogeneity ensures that the formulation preparation procedure is adequate for the study.

Homogeneity of a preclinical dose formulation can be affected by multiple factors, including, but not limited to:

• Batch size or volume

• Mixing methods

• Uniform particle size of the test article

• Vehicle composition.

Homogeneity should be reassessed when there is a significant change in the batch size. For instance, if 100 mL of a formulation are prepared for the first study and 1000 mL are prepared for a subsequent study, homogeneity is performed on both batches. These tests ensure that the formulation process at each scale yields a homogeneous mixture. The need for these two processes is based on mixing techniques, because different methods are used depending on batch size.

Once homogeneity has been established using a specific formulation procedure, it is crucial to maintain consistent methods for preparation throughout the study. Minor changes may affect the ability to prepare and maintain a homogeneous mixture.

Challenges

Challenges with homogeneity can vary depending on the type of formulation. There are multiple types of dose formulations used for in vivo studies, including, solutions, suspensions, and solids as described below:

• Solutions by definition are homogenous because all components dissolve in the solvent. If solubility data in the specified vehicle at the highest specified concentration do not exist, it is crucial to confirm that the dosing formulation is in fact a solution; therefore a homogeneity assessment is required for the preclinical study. This assessment is a regulatory requirement by FDA for preclinical safety studies. It ensures that assumptions based on visual observation or solubility information in another solvent other than the vehicle do not compromise the outcome of an in vivo study.

• Suspensions, where not all components are dissolved, require additional attention to ensure that the components are evenly distributed and remain distributed throughout the course of dosing. This assessment may require extra steps, such as continuous stirring or heating, while dosing. Suspensions that are stored prior to dosing may require extended stirring or heating to ensure homogeneity of the formulation prior to dosing.

• Solid-dose formulations, including gels, creams, and feeds, add additional challenges to establishing and maintaining homogeneity. Adequate mixing and blending are even more important when preparing a solid formulation. Regularity of particle size is key to feed blends because small amounts of test article are being added and mixed to relatively large amounts of feed. Several tactics may be used to improve solid homogeneity, including sieving or grinding of the test material prior to adding to the feeds, or dry blending the test article with a secondary substance prior to addition of feed. Spray-dried techniques used in the manufacturing of marketed products are typically not feasible because of scale and frequency of preparation needed for a preclinical study.

Examples

As noted, assessing homogeneity for preclinical dose formulations is typically assessed by analyzing samples taken from the top, middle, and bottom of a formulation batch using a validated analytical method. Two samples are removed from each stratum, for analysis resulting in a sampling size of n=6. The sample results are compared with standard acceptance criteria for precision (percent relative standard deviation or % RSD). Typical acceptance criteria are featured in Table I.

Table I: Typical acceptance criteria.

On rare occasions, homogeneity assessments fail and it is necessary to modify the procedure for the preparation of a dosing formulation. For example, a feed study being dosed at 1 mg/g produced the following homogeneity results: The variability observed in Table II indicated the possibility that the material was not sufficiently mixed or the test article was not uniform. Homogeneity was improved significantly by grinding the test article in a pestle with a mortar, followed by sieving the material to ensure uniformity prior its addition to feed.

Table II: Homogeneity results. RSD is relative standard deviation

A second example of a failed homogeneity assessment involved a suspension. A test batch was prepared prior to the study to establish an appropriate preparation procedure. Analysis of the test batch indicated that the material was homogeneous. However, analysis of the initial study batch indicated the dose formulation was not homogeneous. Observed differences between the test and study batch were batch size, mixing techniques (related to batch size), and lot of test article.

Following an extensive investigation, it was determined that the lot of test article adversely affected homogeneity. The test articles used in the preparations were sourced from two different manufacturers; the one used for the study batch was not of uniform particle size. To prepare a homogenous dose formulation using the second lot of material required grinding with a mortar in a pestle and then using a sieve to ensure uniform particle size.

In another example, a dose formulation suspension prepared in 0.5% aqueous methylcellulose, required several modifications prior to successfully preparing a homogenous dosing formulation. In this instance, multiple test batches were prepared using various homogenization and sonication times. The final preparation required dry grinding of the test article using a pestle and mortar. The material was subjected to additional grinding after the addition of small volumes of vehicle to form a smooth paste. The remaining volume of vehicle was added and the formulation was mixed for 5 min using a Silverson homogenizer, sonicated for 30 min and then stirred for 1 h. Subjecting the formulation to extensive mixing and sonication were required to prepare a homogenous material.

Conclusion

The preparation of a homogeneous dosing formulation is a critical component of any preclinical study. Homogeneity depends upon various factors, including batch size, mixing and blending techniques, and particle size. Accurately determining homogeneity depends upon standardized sampling techniques and using appropriate analytical methodology.

If possible, the use of test batches prior to study initiation can go far in predetermining homogeneity. If the preparation methodology is appropriate, it can facilitate successful study execution.

Amy Smith is director, and Melissa Whitsel is analytical manager, both with MPI Research; headquarters located at 54943 North Main Street, Mattawan, MI 49071.

References

1. FDA 21 CFR Part 58, Good Laboratory Practice Regulations, Final Rule.

2. M. Whitmire et al., AAPS Jrnl., 12 (4), 628–634 (2010).