pharmaceutics

Article
A Critical Overview of FDA and EMA Statistical Methods to
Compare In Vitro Drug Dissolution Profiles of
Pharmaceutical Products
Jan Muselík 1 , Alena Komersová 2, * , Kateřina Kubová 1 , Kevin Matzick 2 and Barbora Skalická 2

1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Masaryk University, Palackého tř. 1,

612 00 Brno, Czech Republic; [email protected] (J.M.); [email protected] (K.K.)
2 Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573,
532 10 Pardubice, Czech Republic; [email protected] (K.M.);
[email protected] (B.S.)
* Correspondence: [email protected]

Abstract: A drug dissolution profile is one of the most critical dosage form characteristics with
immediate and controlled drug release. Comparing the dissolution profiles of different pharma-
ceutical products plays a key role before starting the bioequivalence or stability studies. General
recommendations for dissolution profile comparison are mentioned by the EMA and FDA guide-
lines. However, neither the EMA nor the FDA provides unambiguous instructions for comparing
the dissolution curves, except for calculating the similarity factor f 2 . In agreement with the EMA
and FDA strategy for comparing the dissolution profiles, this manuscript provides an overview of





 suitable statistical methods (CI derivation for f 2 based on bootstrap, CI derivation for the difference
between reference and test samples, Mahalanobis distance, model-dependent approach and maxi-
Citation: Muselík, J.; Komersová, A.;
Kubová, K.; Matzick, K.; Skalická, B.
mum deviation method), their procedures and limitations. However, usage of statistical approaches
A Critical Overview of FDA and for the above-described methods can be met with difficulties, especially when combined with the
EMA Statistical Methods to Compare requirement of practice for robust and straightforward techniques for data evaluation. Therefore,
In Vitro Drug Dissolution Profiles of the bootstrap to derive the CI for f 2 or CI derivation for the difference between reference and test
Pharmaceutical Products. samples was selected as the method of choice.
Pharmaceutics 2021, 13, 1703.
https://doi.org/10.3390/ Keywords: drug dissolution; dissolution profile comparison; EMA and FDA strategy
pharmaceutics13101703

Academic Editor: Paola Mura

1. Introduction
Received: 9 September 2021
Accepted: 13 October 2021
A dissolution study determines the in vitro release of an active pharmaceutical ingre-
Published: 15 October 2021
dient (API) from the tested dosage form in the prescribed dissolution medium within a
specified time interval [1]. A detailed API dissolution profile resulting from dissolution
Publisher’s Note: MDPI stays neutral
testing represents one of the most essential dosage form characteristics. The dissolution
with regard to jurisdictional claims in
test is routinely used to provide critical in vitro drug release information in pharmaceutical
published maps and institutional affil- development and the quality control process [2,3]. It is applied in the development of
iations. brand-name drugs and generics as well. It plays a crucial role in comparing the dissolution
profiles of different pharmaceutical products prior to starting the bioequivalence studies [3].
The dissolution test is an essential tool in assessing the quality and stability of a medicinal
product [1,4,5]. The primary prerequisite for a qualified estimation of an API release is
Copyright: © 2021 by the authors.
selecting the appropriate dissolution method with distinctive character, which can clearly
Licensee MDPI, Basel, Switzerland.
detect the main differences in the API release from individual dosage forms, ideally under
This article is an open access article
the conditions mimicking those for real drug absorption in the human or animal body [3].
distributed under the terms and The list of dissolution methods for individual APIs registered in the USA is published by
conditions of the Creative Commons the Food and Drug Administration (FDA) on its website [6].
Attribution (CC BY) license (https:// Experimental dissolution data can be used for the prediction of drug release rate
creativecommons.org/licenses/by/ and mechanism from the dosage form. Various mathematical models [7–10] expressing
4.0/). the amount of API released as a function of time are commonly used for processing the

Pharmaceutics 2021, 13, 1703. https://doi.org/10.3390/pharmaceutics13101703 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 1703 2 of 12

dissolution data. Due to the non-linearity of the obtained dissolution curves (except for
the zero-order kinetics), the use of non-linear regression analyses to evaluate the original
measured data should be preferred. The dependency transformation of measured data into
a linear form (e.g., power or logarithmic transformation) causes a change in the deviation
size and the sum of squares of deviations is not minimized by the resulting function.
An important area in dissolution data analyses is the assessment of the similarity
between the dissolution profiles. Several approaches have been developed for comparing
the dissolution profiles. To this aim, the f 2 method proposed by Moore and Flanner [11]
is, due to its simplicity, the metric of choice by both the European Medicines Agency
(EMA) and FDA [12,13]. The f 2 similarity factor is used to assess the global similarity
of the dissolution curves and it does not require any assumption regarding the data-
generating process. If a substantial variation from batch to batch exists, the use of the f 2
point estimate in comparing two drug dissolution profiles is not appropriate [14,15]. To
circumvent this problem, the EMA and FDA guidelines allow other model-dependent
or model-independent methods to be used for the dissolution profile comparison. The
model-dependent methods fit the model represented by some mathematical function
on data, as described above. The similarity region is derived based on a variation of
estimated parameters from the fitted model for test units. The model-independent methods
determine the similarity limits in terms of multivariate statistical distance. This article aims
to provide a detailed overview of the FDA and EMA mentioned methods used to compare
the dissolution profiles, including their procedures and limitations.

2. Dissolution Profile Comparison in the Context of the EMA and FDA Guidelines
The EMA and FDA guidelines [12,13] allow different approaches to be used for dis-
solution profiles comparison. The general recommendation is to use at least 12 units
(e.g., a unit represents, for example, a tablet) of both the test (T) and reference (R) products
to determine the similarity of the dissolution profiles. The dissolution profile similarity
testing can be considered valid only if the dissolution profile has been satisfactorily charac-
terized using a sufficient number of sampling time points. Where more than 85% of the
drug is dissolved (which means the mean percent of drug released for both (R) and (T)
samples) within 15 min, the dissolution profiles may be accepted as similar without further
mathematical evaluation. If the previous requirement is not met, the similarity factor f 2 is,
due to its simplicity, the metric of choice by both the EMA and FDA [12,13]. When the ƒ2
statistic is not suitable, then the EMA guidelines [12] suggest that “( . . . ) the similarity may
be compared using model-dependent methods or model-independent methods ( . . . )” and these al-
ternative methods must be “( . . . ) statistically valid and satisfactorily justified”. Except for the
f 2 statistic, several model-independent approaches have been developed and investigated,
e.g., the Rescigno indices [16], ratio-test approaches [17], methods based on the analysis
of variance (ANOVA) model [18–20], or calculation of the dissolution efficiency based on
the area under the curve [21,22]. Unfortunately, the EMA and FDA guidelines [12,13] are
not clear about the acceptability of the above-mentioned examples of model-independent
methods. The EMA guidelines [12] only state which condition should be generally fulfilled
if some alternative method is used. This condition is that the “( . . . ) similarity acceptance
limits should be pre-defined and justified and not be greater than a 10% difference” and that
“( . . . ) the dissolution variability of the test and reference product should be also similar, however,
a lower variability of the test product may be acceptable”. On the other hand, the FDA guide-
lines [13] are more specific regarding similarity assessment based on model-dependent
methods, i.e., comparison of profiles based on fitted mathematical function(s) to dissolution
profiles. The following parts describe the basic approaches for comparing the dissolution
profiles, an overview of statistical methods and recommendations in the context of the
EMA and FDA guidelines (see Figure 1).
Pharmaceutics 2021, 13, 1703 3 of 12

Figure 1. Schema of the current strategy in a dissolution data comparison by the EMA and
FDA guidelines.

2.1. Similarity Factor f2

A simple model-independent approach uses a similarity factor f 2 to compare the
dissolution profiles [11]. The similarity factor f 2 is a logarithmic reciprocal square root
transformation of the sum of squared differences between the (T) and (R) profiles and it
represents the measurement of similarity in the percentage (%) of dissolution between the
“average” dissolution profiles, i.e.,
 " #−0.5 
 1 n 
f 2 = 50·log 100· 1 + ∑ ( Rt − Tt )2 (1)
 n t =1 

where n is a number of time points and Rt and Tt are the mean percentages of the released
drug from the (R) and (T) products, respectively, at the t time point, 1 ≤ t ≤ n. The
logarithm in Equation (1) is decadic, i.e., with base 10.
The similarity factor reaches values from 0 to 100. The value of 100 indicates identical
dissolution profiles. A negative value negligibly different from value 0 indicates maximal
percentage dissimilarity between the profiles, i.e., 0% for the first product and 100% for
the second product at each non-zero time point. The f 2 value in the range between
50–100 suggests similarity of the dissolution profiles according to the EMA and FDA
guidelines [13,23]. A more precise statement that the value of f 2 has to be strictly greater
than 50 for similarity is provided in the FDA guidelines [13]. Based on f 2 , the dissolution
profiles are indeed considered dissimilar if the mean percentage of the released drug at
each time point differs by at least 10% between the (T) and (R) products. In such a case, f 2
is always less than 50.
The similarity factor f 2 cannot be used on arbitrary dissolution data. According to the
EMA and FDA guidelines [12,13], the following prerequisites must be satisfied:
Pharmaceutics 2021, 13, 1703 4 of 12

(1) the dissolution measurements are made under the same conditions for both products;
(2) a minimum of three-time points (time zero excluded) is considered for both products;
(3) the time points at which the dissolutions are measured are the same for both products;
(4) at least 12 individual dosage units are used for both products;
(5) not more than one mean percentage value is higher than 85% for any of the products;
(6) the coefficient of variation (CV) of either product should be less than 20% at the first
(7) (non-zero) time point and less than 10% at the following time points.
Other approaches to evaluate the similarity of the dissolution profiles have to be used
if at least one prerequisite for the f 2 calculation is not met, e.g., the CV values at some
time points exceed the maximum permitted value. In this respect, the EMA guidelines [12]
prefer the bootstrap method for the construction of the confidence interval (CI) for f 2 (see
Section 2.2). On the contrary, the FDA guidelines [13] prefer using some multivariate
statistical distance (of which the Mahalanobis distance is the most common example),
on the original dissolution data (see Section 2.4) or on the estimated parameters from
regression models fitted to the original dissolution data (see Section 2.5).
It should be mentioned that the non-fulfillment of at least one prerequisite for using
the f 2 does not seem to be the only problem. The dissolution profile similarity can be
concluded with the f 2 even if the difference between the (R) and (T) samples in the mean
percentage of the drug dissolved at some time point (or points) is greater than 10%. It
is inconsistent with the similarity acceptance limits, which should “( . . . ) not be greater
than a 10% difference” as stated in the EMA guidelines [12], which refer to a 10% difference
in individual time points [23]. Therefore, alternative statistical approaches to assess the
similarity of dissolution profiles between the (T) and (R) products should be used in
conjunction with the f 2 to comply with the maximally 10% difference criterion. Such
criterion can be an estimation of CI for the difference between the (T) and (R) products at
each time point where all CIs should lie entirely within the similarity acceptance limits (see
Section 2.3).
According to the FDA guidelines [13], it is also possible to use the difference factor
f 1 for the dissolution profile comparison. The difference factor is a sum of the absolute
values for the differences between the (T) product and (R) products relative to the sum
of the mean percentage of the released drug from the (R) product. This approach to
assessing the dissolution profiles’ similarity is based on the same principle as when using
the similarity factor. A calculated value of f 1 in the range of 0–15 suggests the dissolution
profiles similarity between the (T) and (R) products. The value 0 corresponds to identical
dissolution profiles (including the special case when both products have identically zero
mean percentage of the released drug at each time point). Interestingly, the f 1 can have
a theoretically unbounded upper limit if the (T) product is approaching the maximal
dissolution (~100%) at each non-zero time point and simultaneously the (R) product
is approaching minimal dissolution (~0%) at each non-zero time point. Details on the
calculation of the difference factor are given in the literature [11,13].
The difference factor f 1 and similarity factor f 2 represent a simple and frequently used
tool for comparing in vitro dissolution profiles. The use of this approach does not require
deep knowledge in the field of statistics nor special software. It is the method of the first
choice if the prerequisites mentioned above are satisfied. If at least one prerequisite for
the f 2 calculation is not met (e.g., CV values at some time points are higher than the maxi-
mum acceptable value), the EMA and FDA guidelines recommend using some alternative
approach to evaluate the similarity of the dissolution profiles. The EMA guidelines [24]
prefer the construction of CI for f 2 based on the bootstrap method (Section 2.2). The FDA
guidelines [13] prefer to use some multivariate statistical distance, e.g., the Mahalanobis
distance (see Section 2.4.) or comparison of the dissolution profiles based on the regression
parameters from the regression analysis of the dissolution data (see Section 2.5).
Pharmaceutics 2021, 13, 1703 5 of 12

2.2. CI Derivation for f2 Based on Bootstrap

As stated previously, the bootstrap method for deriving CI for f 2 is recommended by
EMA [23] if the f 2 statistic alone is not suitable. As described in Islam and Begum [14],
bootstrap “( . . . ) is computationally-intensive approach to statistical inference ( . . . ) based on the
sampling distribution of a statistic obtained by resampling from the data with replacement”, which
enables the deriving of “( . . . ) an exact sampling distribution of a statistic of interest”. The
statistic of interest is f2 and based on knowledge of its sampling distribution, the bootstrap
CI for f 2 can be obtained. For dissolution data, including 12 profiles for both products
(which is the minimum requirement on a number of profiles for using f 2 ), it is preferable to
use the so-called non-parametric bootstrap to construct CI for f 2 [14].
Mendyk et al. [24] created statistical open-source software “PhEq_bootstrap version
1.2” to assess the dissolution profiles’ similarity, whose principle is based on the bootstrap
methods under the EMA regulation.
An example of how to proceed in the bootstrap method is as follows [25]:
(1) generate n bootstrap samples (e.g., 5000) by re-sampling from the original dissolution
data;
(2) calculate f 2 for each of the n bootstrap samples, i.e., there are n new f 2 values;
(3) derive CI (lower and upper bounds) using the appropriate approach.
Regarding point (1), the test (reference) profiles in the bootstrap sample are re-sampled
from the test (reference) profiles in the original data. Regarding point (3), Islam and
Begum [14] concluded that the bias-corrected and -accelerated approach for calculating
the percentiles leading to lower and upper bounds of bootstrap CI performs best. The
dissolution profile similarity based on bootstrap CI for f 2 is concluded when the lower limit
of the bootstrap CI is entirely above 50 [23]. It also meets the more precise requirement
found in the EMA guidelines [12], namely, that “( . . . ) f 2 value between 50 and 100 ( . . . )”
for similarity means that f 2 has to be greater than 50.
Islam and Begum [14] mention that a two-sided 90% bootstrap CI for f 2 , preferably a
two-sided 95% bootstrap CI, should be used.
The bootstrap method allows researchers to perform the comparison of highly variable
data sets and obtains a reliable result due to limiting the impact of outliers. As mentioned
above, this approach is supported by EMA in cases when the prerequisites for f 2 calculation
are not met and the use of f 2 statistics alone is not suitable. The CI derivation for f 2
based on bootstrap requires a more profound knowledge of statistics in comparison to
the conventional approach (the f 2 factor calculation), but the use of special software (e.g.,
statistical open-source software created by Mendyk [24]) can facilitate the calculation.

2.3. CI Derivation for the Difference between Reference and Test Samples
Based on a statement in the EMA guidelines [12] that “( . . . ) similarity may be compared
using ( . . . ) the percentage dissolved at different time points ( . . . )”, another simple approach in
statistical comparison of dissolution profiles is to calculate the CI for the difference between
the (T) and (R) sample with respect to mean percentage of the released drug at each time
point separately. Taking into account the statement in the EMA guidelines [12] that the
difference should “( . . . ) not be greater than a 10% ( . . . )”, the dissolution profiles can be
considered similar if the CI for the estimated difference between the (T) and (R) sample at
each time point lies entirely within the interval (−10%, +10%). The lower and upper bound
of the CI can be estimated according to the following expression:
s 
1 1
Rt − Tt ± Q p + s2 (2)
n T,t n R,t t

where Rt and Tt are the mean percentage of the released drug for the (R) and (T) samples,
respectively, at the t time point; Qp is the p-quantile of the relevant probability distribution
where number p is chosen from the interval (0,1) to obtain target confidence level for CI;
nT,t and nR,t denote the number of measurements for the (R) and (T) samples, respectively,
Pharmaceutics 2021, 13, 1703 6 of 12

at t-the time point; st 2 is the pooled variance that estimates the population variance using
the weighted average of variances sR,t 2 and sT,t 2 from (R) and (T) samples, respectively, at
the t-the time point. Thus, the pooled estimate of variance is expressed by the equation

(n R,t − 1)s2R,t + (n T,t − 1)s2T,t

s2t = (3)
n R,t + n T,t − 2

In principle, Equation (2) is based on rewriting the test statistic for the two-sample
t-test. In this case, quantile Qp in (2) is the p-quantile of t-distribution with nT,t + nR,t − 2
degrees of freedom (a number of observations for the (T) and (R) products minus two at
t-the time point) if the following assumptions are met:
• the observations within each sample and between samples are independent;
• the two samples follow normal distributions;
• the two samples being compared have the same variance.
Usually, a two-sided 95% CI for the difference is considered, corresponding to p = 0.975
for Qp .
The shortcoming of the two-sample t-test can be seen, for instance, in ignoring the
dependence in the measured dissolutions of the same profile between different time points.
The quantile resulting from the distribution of the test statistic for the multivariate two-
sample Hotelling’s T2 -test can be used to overcome this limitation. In the literature, the
dissolution profile comparison using T2 -statistics has been described in detail [26].

2.4. Mahalanobis Distance

A potential alternative to the similarity factor f 2 can be the Mahalanobis distance
(MD), the most common example of multivariate statistical distance.
In general, the Mahalanobis distance [27] is the distance between a point and distribu-
tion (not between two distinct points). The MD is equal to zero if the point is at the mean
of the distribution and increases if the point moves away from the mean.
The assessment of similarity of the dissolution profiles between the (R) and (T) prod-
ucts using MD is based on the comparison of the vectors of the mean values of the drug
released amount from the (R) and (T) products at given times. The MD for dissolution data
of the (R) and (T) products is calculated according to
q
MD = ( T − R ) T S −1 ( T − R ) (4)

where T and R is the vector of the mean percentage values of the released drug at given
times for the (T) and (R) product, respectively; (T − R)T is the transposition of the vector
of the differences (T − R); S−1 is the (existing) inverse matrix to the empirical covariance
matrix S. It should be noted that, in practical calculations, the empirical covariance matrix
S is based on the pooling covariance matrix for the (R) product with the covariance matrix
for the (T) product. In addition, suppose there are n time points at which the dissolutions
are measured. In that case, the vector of means and differences between the means has
n components and the empirical covariance matrix is an n × n matrix. The empirical
covariance matrix accounts for the dependencies in the dissolutions between different time
points. The multiplication of vectors with the inverse of covariance matrix in Equation (4)
gives MD as a scalar with non-negative value.
For the calculation of MD, the PC software (e.g., R statistical software, Python™)
which has been implemented Equation (4) can be used. If the covariance matrix S is the
identity matrix, then its inverse S−1 is the identity matrix as well and the MD reduces to
the Euclidean distance. For the estimated value of MD, 90% or 95% CI can be calculated.
The key and critical step for assessing the profile similarity based on the MD value is the
determination of the equivalence margin θ (similarity threshold). For the calculation of
θ, the vectors of differences (T − R) and (T − R)T should be the vectors with the tens to
comply with the condition in the EMA guidelines, i.e., that the difference should “( . . . )
Pharmaceutics 2021, 13, 1703 7 of 12

not be greater than a 10% difference” [12] between the (T) and (R) products at each time point.
The inverse matrix S−1 in Equation (4) is calculated from the pooled covariance matrix S
derived from the measured dissolution profiles. The dissolution profiles can be considered
similar if the upper limit of CI for MD is lower than the value of equivalence margin θ.
In other words, the similarity is concluded if CI for MD is fully within the interval [0, θ)
where the lower limit of the interval uses the fact that MD defined in Equation (4) has
the minimum value zero. It should be mentioned that some authors share the view that
the equivalence margin should be a predefined fixed number [28,29] in comparison to
the margin based on Equation (4), which is, apart from the chosen vectors of differences
(T − R) and (T − R)T , influenced by the inverse of the empirical covariance matrix S. The
current multivariate equivalence procedures based on MD were described in detail by
Hoffelder [30]. Hoffelder [30] did not discuss only the use of Euclidean and MD in the
context of the dissolution profile comparison. Moreover, he mentioned the critical moments
on the MD in the current literature.
The EMA published a document concerning the adequacy of the MD to assess the
comparability of drug dissolution profiles in 2018 [23]. In this document, the EMA prefers
the bootstrap methodology to derive CI for f 2 and does not recommend the MD for the
dissolution profile comparison. The uncertainties mentioned above can affect this EMA
recommendation, mainly because the equivalence limit is not given and the definition of
profile similarity can vary among the dissolution datasets.

2.5. Model-Dependent Approach, Maximum Deviation Method

The similarity of the dissolution profiles in cases when the f 2 metric is not suitable,
can be also assessed using the model-dependent approach. This approach is based on
fitting a regression model to the dissolution profiles (the most frequently used models are
described in Table 1), the estimation of the model parameters and a comparison of the
model parameters between the products for the similarity assessment. The fundamental
steps in the dissolution profile regression analysis are summarized in detail in the article
recently published by Muselík et al. [31]. For the dissolution profile comparison based
on the model-dependent approach, the FDA guidelines [13] recommend to fit the model
with no more than three parameters to each individual profile, apply some multivariate
statistical distance (MSD) on the estimated parameters, derive 90% CI or 90% confidence
region (CR) for MSD and, if the 90% CI or 90% CR lies entirely within the similarity region,
then the similarity of the dissolution profiles is concluded. The EMA guidelines [12] are
not specific regarding the similarity assessment, except for possible acceptability of MSD
applied on estimated parameters from the Weibull model.

Table 1. Frequently used mathematical models to describe the drug dissolution profiles.

Model Equation Unit of (Rate) Constant

Zero-order Mt = k 0 t + b amount of drug time−1 (e.g., mg h−1 )
The first-order [32] Mt = M∞  (1 − exp(−
 k1 t))  time−1 (e.g., h−1 )
Weibull [33,34] Mt = M∞ 1 − exp −k w t β time−β
Korsmeyer–Peppas [35–39] Mt
M∞ = k KP tn time−n
Mt
√
Higuchi [40–47] M∞ = kH t time−1/2
 1 3
Hixson–Crowell [48,49] 0
Mt = M∞ 3
− k HC t drug amount1/3 time−1 (e.g., mg1/3 h−1 )

Hopfenberg [50] Mt
M∞ = 1 − (1 − k HP t)N time−1

Mt is the amount of drug released in the time t, k0 is the zero-order release rate constant,
parameter b represents a non-zero dissolved drug amount Mt in the dissolution medium
in time t = 0, M∞ is the maximum amount of drug which can be released from a dosage
form in infinite time, k1 is the first-order release rate constant, kw is the constant of Weibull
model, the parameter β (Weibull model) characterizes the shape of the exponential curve,
Pharmaceutics 2021, 13, 1703 8 of 12

kKP is the constant of Korsmeyer–Peppas model, kH is the constant of Higuchi model, M’t
is the drug amount in a dosage form in the time t, kHC is the constant of Hixson–Crowell
model and kHP is the constant of Hopfenberg model.
However, based on statements in the FDA guidelines [13], it is unknown how to
derive the similarity region for estimated model parameters. Moreover, there is also a
question why the FDA guidelines [13] recommend using only point estimates of the model
parameters for similarity assessment based on MSD. Still, the standard errors of point
estimates are not considered.
Regarding the choice of similarity region for model parameters, the solution could be
to fit all dissolution profiles of the (R) and (T) products to the same regression model (e.g.,
using OriginPro® 9, GraphPad Prism® 7). The same regression model clearly enables the
comparison of the corresponding regression parameters between products. The regression
model should be optimally fitted to all dissolution profiles of the (T) and (R) products at
the same time points in order to use as much information as possible in the fitting process.
The model should also be considered in such a form that the difference between the (T) and
(R) products can be expressed. The similarity criterion for the difference in the estimated
values of the model parameters between the products is then formulated based on the
difference between the function values of the chosen model for the (R) and (T) profile.
Paraphrasing the EMA guidelines [12,23], the difference in the percentage of the released
drug between the (T) and (R) profiles should not be greater than 10% in the absolute value,
i.e., |T(t) − R(t)| ≤ 10, for each time point t from a specific time range. In this manner, the
acceptable difference between the estimated parameters is defined as a restriction on the
difference between the function values at each time point.
Regarding the inclusion of the standard error, (1 − α), 100% CI for the difference
between the estimated parameters of the (T) and (R) products mentioned above can be
estimated according to the
difference between parameters” ± tv,1-α * “standard error of the difference” (5)

where tv,1–α is a (1 − α)-quantile of the t-distribution with v = nT,t + nR,t − 2 degrees of

freedom (reflecting the total number of observations minus 2) and α is from the interval
(0,1). The value of α is chosen in such a way to obtain the required confidence level for
CI (usually 1 − α = 0.975 to obtain two-sided 95% CI for the difference in Equation (5)).
The dissolution profiles of the (R) and (T) products can be considered similar if the CI
for the difference between the (R) and (T) samples based on Equation (5) is fully within
the range, reflecting, maximally, a 10% difference between the dissolution profiles, as
described above.
An example can be given for the zero-order kinetic model without the intercept term,
i.e., with T(t) = kT t and R(t) = kR t (where kT and kR are the release constants for the (T) and
(R) products, respectively). The criterion for the difference between the parameters is |kT
− kR | ≤ 10/t. This forms the similarity region [−10/t,+10/t] in which the CI for difference
kT − kR must lie entirely. The lower and upper limits of the similarity region depend on
time t from a specific time range and the time unit is the same as the time unit used for
fitting the regression model.
If the intercept term (“burst effect”) is present, then CR (typically 90% or 95%) is
simultaneously created for the intercept term and slope term. Consequently, the condition
on similarity must be fulfilled for all combinations of values of both parameters from
simultaneous CR. For example, considering a zero-order kinetic model with the intercept
term, i.e., T(t) = k0T + kT t and R(t) = k0R + kR t, the condition on similarity is |(k0T − k0R )
+ (kT − kR ) t| ≤ 10 with time t chosen from a certain time range. The condition must be
satisfied for all combinations of k0T , kT , k0R and kR from the derived simultaneous CR.
However, considering the intercept term rather leads to the situation where the same
regression model is fitted separately to the (T) and (R) profiles. Such an approach of
comparison parameters between two simple linear regression models and derivation of
simultaneous CR was described [32].
Pharmaceutics 2021, 13, 1703 9 of 12

It should be noted that Equation (5) is the most suitable for zero-order, Korsmeyer–
Peppas, or the Higuchi model. In these models, the absolute difference between the
parameters can be easily derived for a conclusion regarding similarity using the same
model for all dissolution profiles of the (T) and (R) products simultaneously.
The expression of the similarity criterion for model parameters becomes more compli-
cated if the estimated parameter of the used model is in the argument of the non-linear
function. For example, in the case of the first-order kinetic model with T(t) = M∞ (1 −
exp(–kT t)) and R(t) = M∞ (1 − exp(–kR t)), an approximation approach has to be used to
derive the similarity criterion for the difference kT − kR . Supposing that the maximum
amount M∞ of the released drug is the same for both products, the possible choice is to
use the first-order Taylor polynomial leading to |M∞ (kT − kR ) t| ≤ 10 and the similarity
region (−10/(M∞ t), +10/(M∞ t)). This region depends not only on time t but also on the
maximal release M∞ . In this case, the corresponding CI for the difference kT − kR can be
derived based on Equation (5) using the estimates and their standard errors obtained from
the model fitted separately to all dissolution profiles of the (T) product and separately to
all dissolution profiles of the (R) product. The disadvantage is that the similarity region for
the difference kT − kR is not exactly expressed. Moreover, the derivation of simultaneous
CR for the non-linear model is more complicated [32]. This difficulty is even higher if
the parameters kT and kR are derived from separate (but the same) non-linear regression
models.
To avoid complicated derivations for both the similarity region and CR on the “case-
by-case basis”, an alternative method was proposed. Such method is called the maximum
deviation (MAXDEV) method [33] and it is based on the assessment of the difference

d∞ = maxt∈ M | T (t, β T ) − R(t, β R )| (6)

where T(t, βT ) and R(t, βR ) are parametric regression curves fitted to the samples of the
(T) and (R) products, respectively; βT and βR are vectors of corresponding regression
parameters; t denotes time from the interval M. Null hypothesis (H0 , Equation (7)) against
the alternative hypothesis (H1 , Equation (8)) is formulated as

H0 : maxt∈ M | T (t, β T ) − R(t, β R )| ≥ ∆ (7)

vs. H1 : maxt∈ M | T (t, β T ) − R(t, β R )| < ∆ (8)

where ∆ is a pre-specified positive number. In the context of the statement from the EMA
guidelines [12] related to tolerating, maximally, a 10% difference at each time point, the
choice is ∆ = 10. The formulation of H0 is that the dissolution profiles are not similar
with respect to the chosen models for the (T) and (R) samples as the maximal absolute
difference exceeds ∆. To better approximate the distribution of d∞ under H0 , the CI for d∞
is estimated based on the parametric bootstrap with a restriction on the estimated model
parameters. If such CI for d∞ lies entirely in the interval [0, ∆), where the lower limit of
the interval results from the fact that d∞ is defined as a non-negative value, the similarity
between the dissolution profiles is concluded. The MAXDEV method has the advantage
that the variability, time dependence between the measured dissolutions and the shapes of
the dissolution profiles are simultaneously taken into account. This methodology seems to
have the ability to overcome the issue when the fitted regression model is different for the
(T) profiles and for the (R) profiles, although the authors demonstrated the performance of
the MAXDEV method only on the same model chosen for both products. As the bootstrap
is parametric, the bootstrap samples of dissolution profiles are based on bootstrapping
from some parametric distribution. Specifically, the MAXDEV method bootstraps residuals
from a normal distribution with zero mean and covariance structure which is estimated
from the residuals resulting from the fitted models applied on the original dissolution data.
Based on the bootstrap samples, the model parameters from the originally fitted models
are estimated and the value of d∞ is calculated. Finally, the percentile method is used for
finding suitable quantiles of the estimated distribution of d∞ to obtain the bootstrap CI
Pharmaceutics 2021, 13, 1703 10 of 12

of the requested confidence level for d∞ . Moellenhoff et al. [33] tested the performance
of the MAXDEV method on three real datasets and an artificial dataset. Based on the
artificial dataset, it is shown that the type 1 error probability (T1EP) is controlled at 5%
compared to the bootstrap method for construction of 90% CI for f 2 (criterion for similarity:
the lower limit of 90% CI has to be higher than 50) where T1EP is inflated (~55%). The
statement about T1EP is made for ∆ = 10 in H0 for d∞ , as the authors are probably aware.
It is compliant with the condition that the difference in dissolutions between the (T) and
(R) products “( . . . ) should not be greater than a 10%” [12]. The authors believe that the
discrepancy in T1EP may be caused by the condition that d∞ needs to have the difference
“uniformly” less than 10% at each time point to conclude similarity, while f 2 can assess
similarity even if the difference is at least 10% at some time point (see also the brief mention
in Section 2.1). The statistical power for the MAXDEV method was higher than 80% for
values d∞ ≤ 8 and then declined (e.g., the power for d∞ = 9 was around 30%). The power
for the bootstrap f 2 was generally higher but at the cost of inflated T1EP and, as the authors
mention, such inflation” ( . . . ) is unacceptable from the regulatory point of view”.
Using the model-dependent approach requires (i) the knowledge of the mathematical
models suitable for describing the different types of dissolution profiles, (ii) the knowledge
of the principles of non-linear regression analysis and (iii) suitable software. The critical
point of this approach is the expression of the similarity criterion for model parameters,
especially if the estimated parameter is in the argument of the non-linear function. The
MAXDEV method offers the possibility to avoid complicated derivations for both the
similarity region and CR. Moreover, this methodology’s advantage is it overcomes the
issue when the fitted regression model is different for the (T) and the (R) profiles.

3. Conclusions
The release profiles evaluation very often involves the comparison of dissolution
profiles (e.g., comparing generics with brand-name medicines, stability testing of pharma-
ceutical products). Several different methods suitable for comparing dissolution profiles
are described in the literature. This article aims to provide an overview of the approaches
that are supported by regulatory authorities. According to the FDA and EMA guidelines,
the primary approach in comparing dissolution profiles is the similarity factor f 2 . In cases
when the mean percentage of the released drug is higher than 85% within 15 min for both
products, the similarity factor f 2 does not need to be used and the profiles are automatically
considered similar without any further mathematical evaluation. In other cases, either
the similarity factor f 2 (if all of its prerequisites are met) or some alternative statistical
methods (if at least one prerequisite for using f 2 is violated) are considered. In the context
of comparing the dissolution profiles, some approaches described by EMA and FDA (e.g.,
Mahalanobis distance, model-dependent approach) are burdened with some limitations
(e.g., the guidelines do not mention the similarity criteria). On the other hand, the bootstrap
for the derivation of CI for f 2 is an acceptable approach with clearly set criteria in statistical
comparison of in vitro dissolution profiles [23]. An acceptable approach could also be the
comparison of the difference between the (T) and (R) samples concerning the percentage
dissolved at different time points [12] based on the calculation of CI and its comparison
with pre-defined limits. Based on the discussion above, the CI derivation for f 2 based
on the bootstrap and CI derivation for the difference between reference and test samples
could be considered methods of choice. These two methods meet the requirements of the
regulatory authorities, overcome the difficulties in statistical evaluation, are robust and
straightforward for routine use in practice and allow assessment of the different types of
dissolution data.
Pharmaceutics 2021, 13, 1703 11 of 12

Author Contributions: Conceptualization, A.K. and J.M.; data curation, A.K. and J.M.; methodology,
A.K., J.M., K.K., K.M. and B.S.; supervision, A.K. and J.M.; writing—original draft, A.K. and J.M.;
writing—review and editing, K.K., K.M. and B.S. All authors will be informed about each step of
manuscript processing, including submission, revision, revision reminder, etc., via emails from our
system or assigned Assistant Editor. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by the Ministry of Agriculture under the project CZ QK1810221,
by Masaryk University (project MUNI/A/1574/2020) and by the Ministry of Education, Youth and
Sports of the Czech Republic under the project SGS 2021 006.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: A full list of references is compiled and attached to this manuscript.
Acknowledgments: This work was supported by the Ministry of Agriculture of the Czech Republic
under the project CZ QK1810221, by Masaryk University (project MUNI/A/1574/2020) and by the
Ministry of Education, Youth and Sports of the Czech Republic under the project SGS 2021 006. The
authors also thank SOTAX Pharmaceutical Testing s. r. o. and SPS Pharma Services for support and
cooperation.
Conflicts of Interest: The authors declare no conflict of interest.

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