Our Laboratory Testing Blog

Measuring Reproducibility in an In Vitro Release Rate Testing Method

Tweet

 

A performance test for topical drug products must have the ability to measure drug release from the finished dosage form.  It must be reproducible and reliable, and although it is not a measure of bioavailability, the performance test must be capable of detecting changes in drug release characteristics from the finished product.

Product performance tests can serve many useful purposes in product development and in post-approval drug product monitoring.  It must provide assurance of equivalent performance for products that have undergone post-approval raw material changes, relocation or change in manufacturing site, and other changes as detailed in the FDA Guidance for Industry—SUPAC-SS.

Reproducibility of a developed In Vitro testing method (IVRT) can be demonstrated by measuring inter-day and intra-day precision.  To begin, at least 6 Franz Cells should be loaded with the same formulation on the same day (intra) and on three different days (inter).  

Inter-day precision is demonstrated by measuring the release rates of the same formulation (same lot) on three different days.  Each day, the release rate must meet intra-day precision. The release rates from all three days must be less than 15.0% RSD.

Intra-day precision is demonstrated by computing the variability between the release rate from 6 cells of the same formulation (same lot) on the same day.  The RSD of the mean iv vitro release rate must be less than 15.0%.  All cells must show linearity greater than R² of 0.9

The method should also be able to distinguish concentration differences in the same formula. Release rate testing should be performed with samples containing 50%, 100% and 200% label claim of the active. The different formulations are expected to afford release rates difference at least larger than intra-day precision.

According to Higuchi's equation for the suspension, the release rate should follow a square-root relationship with the amount of drug present in the vehicle.  For example, a two-fold increase in drug concentration should result in an √2=1.4 increase in slope.  However, if the drug is fully dissolved from solution, the release rate should be directly proportional to the amount of drug (i.e. a two-fold increase in drug concentration should double the slope).

This validation study will be able to aid in the determination of lot-to-lot and processing variability of the formulation and will provide information to support an in vitro release testing specification of release rate determination using the slope of the linear regression.

A sensitivity experiment should be conducted using two similar Franz cell apparatus (6 cells x 6 cells) in which three strengths of the formulation at 50%, 100% and 200% of label claim are applied in replicates on each Franz Cell apparatus.  To ensure an unbiased comparison, the sample positions of the sensitivity tests within the sets of each 6 vessels should be randomized.

The individual release rate, mean and RSD of the release rate for the six separate samples should then be calculated and reported. The RSD of the release rate for the six separate samples should be equal to or less than 15.0%.  A Comparison of the absolute difference in mean slope (release rate) between formulations of different strengths can then be completed. The method is considered sensitive if the absolute difference is higher than their respective SD values (intra-day precision).

For product sameness testing, the FDA SUPAC-SS Guidance states that the products to be compared should be of comparable age and rates should be determined on the same day, under the same conditions.  To ensure an unbiased comparison, sensitivity test samples within the bank of Franz cells should be randomized.  The test and reference products in each run should be randomized or pre-assigned in a mix arrangement to ensure unbiased comparison.

The slope comparison test recommended by the FDA should be performed and provides the evidence of the reproducibility of the method developed in this study.  The comparison formulations should be applied on 12 cells: 6 cells of the “Reference” formulation and 6 cells of the “Test” formulation and samples collected at different time points.  The Release rates (slope) from six cells of T- product, and the other six cells of the R-product are obtained.  A 90% Confidence Interval (CI) for the ratio (T/R) of median release rates must be computed.  The standard criteria for consideration of product sameness: Test and Reference products are considered to be “same” if the 90% CI falls within the limits of 75% - 133.3%.

Here is how the calculations are performed:

1. Generate a table with seven rows and seven columns. List the reference slopes (R) across the first row and test slopes down the first column of the table.  Compute the individual T/R ratios (36) between each test slope and each reference slope and enter the corresponding values in the table.

2. Rank these 36 T/R ratios from lowest to highest.

3. The 8th and 29th ordered ratios represent the lower and the upper limits of the 90% CI.

The standard criteria for consideration of product sameness: Test and Reference products are considered to be “same” if the 90% CI falls within the limits of 75% - 133.3%.

Note: If the data for a single cell is missing for one of the lots, there would be 5×6=30 individual T/R ratios and the limits of 90% CI would be sixth and twenty-fifth order individual T/R ratios.  If the data is missing for more than one cell, the correct computation should be determined.

If a second stage of in vitro testing is required, then two additional in vitro release testing runs of twelve cells each (six cells per lot) should be performed that will yield 12 additional slopes for each lot or 18 in all (including the first stage results).  A 90% Confidence Interval for the ratio (T/R) of medium release rates is computed using all 18 slopes.  All 324 (18×18) individual ratios are obtained and ranked from lowest to highest.  The 110th and 215th ordered ratios represent the lower and the upper limits of the 90% CI.  The acceptance critera is set as: the Test and Reference products are considered to be “same” if the 90% Confidence Interval falls within the limits of 75%-133.33%.