Use of Enzymes for dissolution of Gelatin capsules

Method development & Validation: Key Elements

Pearl Pereira Nambiar/ Dec 2022

 The Cross-linking phenomena
• Gelatin is a water-soluble protein material

• Cross-linking is expected for capsule drug products filled in hard or soft gelatin shells

• Cross-linking of gelatin is an irreversible alteration of capsule shell structure

• Cross-linking will eventually render the gelatin insoluble in aqueous solvents

• This results in prolonged capsule disintegration time and subsequent slow-down of

the drug-product dissolution rate

• Degree of cross-linking is not uniform causing high variability in dissolution results

 Common causes of cross-linking
• Aldehydes present in active pharmaceutical ingredients, excipients, packaging materials, or
degradants formed in situ during storage (e.g., polyethylene glycols that may auto-oxidize to form
aldehydes)
• High humidity
• Substances that facilitate a cross-linking reaction
• Substances that promote decomposition of stabilizers used in excipients such as
hexamethylenetetramine in corn starch resulting in the formation of ammonia and formaldehyde
• Rayon coilers that contain an aldehyde functional group (furfural)
• Polyethylene glycols that may auto-oxidize forming aldehydes
• UV light, especially in the presence of high temperature and humidity
• Heat, which can catalyze aldehyde formation
 How to identify dissolution failure due to cross-
linking
• The capsules swell, become
deformed, but do not open. A
thin gelatinous mass can be
seen around the capsule
Visual
• Instrumental techniques like
FTIR, DSC, NMR etc. may be
used to detect molecular level
changes induced in the gelatin
shell due to cross-linking
Instrumental
• Capsule switching test:
Transfer the contents of the
cross-linked capsules into
fresh capsules and run the
dissolution test again to
determine if the shells are the
cause of slowed dissolution.
Experimental
 Dissolution failure due to cross-linking
• Significant change?
• The in vitro rate of dissolution of hard and soft gelatin capsules decreased by cross-
linking and may not meet the dissolution specification

• However, failure to meet dissolution specification due to cross linking should not be
considered as ‘significant change’ as in most cases cross-linking does not affect in
vivo behavior

• Enzymes, bile salts etc., promote the opening of the capsule shell in vivo

• The impact of cross-linking on bioavailability is not significant if it can be shown that

the drug release is overcome by addition of enzymes to the dissolution medium.
 Pharmacopoeial provisions
• The United States Pharmacopoeia allows use of enzymes in dissolution media when
gelatin capsules do not conform to dissolution specifications due to cross linking

• Other compendia such as the European Pharmacopoeia allow for the use of
enzymes when justified and authorized by the competent authority (Ph. Eur.
Chapter 5.17),

• The Japanese Pharmacopoeia does not accept addition of enzymes to overcome

cross-linking, but rather recommends the use of succinated gelatin, which is does not
cross-link, for the capsule shell.
 Selection of Enzymes
The enzyme used is selected based on the pH of the dissolution medium

• Dissolution Medium with • Dissolution Medium with pH • Dissolution Medium with

>4.0 and <6.8 pH ≥6.8
pH ≤4.0
• Amount: A quantity of papain that results
• Amount: A quantity of pepsin in an activity of not more than 550,000
• Amount: A quantity of
that results in an activity of units/L of dissolution medium, or a pancreatin that results in a
• quantity of bromelain that results in an protease activity of not more
not more than 750,000 units/L activity of not more than 30 gelatin-
of dissolution medium digesting units (GDU)/L of dissolution than 2000 units/L of
medium dissolution medium

Papain or
Pepsin Pancreatin
Bromelain
Enzyme activity and surfactants in dissolution
medium
• Some surfactants may inhibit enzymatic activity because of
denaturation of the enzyme or by competition for the
substrate.
• Enzyme-surfactant interaction is influenced by so many
variables like enzyme, the solvent, the pH of the medium,
and the characteristics of the enzyme and the outcome
cannot be easily predicted.
• If the loss of enzyme activity is clearly observed, a pre-
treatment step can be applied.
• The volume and time period for the pretreatment step needs to
be determined in a case-by-case approach.
 Pretreatment step
• In this step, the dissolution test for Tier 2 is run with the medium with no surfactants but with the
appropriate enzyme for a short period of time. After that the surfactant is added to the medium and the
dissolution is run for the duration of the test

• The stepwise addition of enzyme and surfactant enables both agents to take effect individually and
sequentially in the dissolution medium.

• The enzyme digests the cross-linked gelatin capsule shell at the beginning to ensure the opening of the
capsule shell

• The addition of surfactant or solubility enhancer afterwards increases drug solubility and/or wettability

• This time is included in the total time of the test. If the time specified in the dissolution procedure is 30
min and you use 15 min in the pretreatment step, you have only 15 min more with the medium containing
surfactant
 Examples of dissolutions with Pretreatment step
• Dutasteride soft gelatin capsules and Ziprasidone HCl capsules are some examples of dissolution
procedure with pretreatment step from the Dissolution procedures listed in the FDA recommended dissolution-
methods database.

• It can also be seen that pretreatment step is not required for Enzalutamide capsules where the dissolution
medium contains a surfactant (CTAB, cationic)
 Method development and Validation
• The development of any dissolution method for gelatin capsules is done with and without the enzyme evaluating
the type of enzyme, filtration procedure, specificity etc.

• To develop and validate the procedure using the enzyme you need to force the presence of cross-linking in your
samples by one of the following options:

1) exposing the gelatin capsules to formaldehyde vapor

2) exposing the gelatin capsules to high humidity and high temperature or

3) filling the gelatin capsules with an excipient spiked with known amounts of formaldehyde

• The validation of the dissolution method is done in just one report for the method with and without the enzyme
including the pretreatment step if applicable

• The final dissolution method for gelatin capsules must have well described procedures with and without
enzymes with the analysts well trained to detect the presence of cross-linking

• Enzymes at concentrations higher than those stated in USP 711 should be investigated and properly justified
 Routine dissolution testing
• The addition of enzymes to the dissolution medium can be done at any stage in the product lifetime (batch
release, stability studies, etc.) but only if there is evidence for the presence of cross-linking in the
gelatin.

• If the noncompliance is due to other reasons, the addition of enzymes is not appropriate.

• The basic composition of dissolution media for Tier 1 and Tier 2 are the same and the only role of the enzyme,
added to the medium in Tier 2, is to digest the cross-linked gelatin, allowing the contents of the capsule to
be released into the dissolution medium

• If the drug product fails at S1 or L1 due to cross-linking, proceed to Tier 2 stage with the addition of
appropriate enzyme to the dissolution medium

• Cross-linking is an irreversible process, hence once observed would continue during storage and may
largely affect the outcome of in vitro dissolution tests
 References
• <711> Dissolution. In: The United States Pharmacopeia and National Formulary, Rockville, MD; USP NF 2022 Issue 1 – Online

• <1094> Capsules—dissolution testing and related quality attributes. In: The United States Pharmacopeia and National
Formulary, Rockville, MD; USP NF 2022 Issue 3 – Online

• https://www.fda.gov/drugs/drug-approvals-and-databases/dissolution-methods-database

• Maria L Guzman, Margareth R Marques, Maria E Olivera ME, Erika S Stippler, Enzymatic activity in the presence of
surfactants commonly used in dissolution media, Part 1: Pepsin, Results in Pharma Sciences, Volume 6, 2016, Pages 15-19, ISSN
2211-2863, https://doi.org/10.1016/j.rinphs.2016.02.002.

• Marques MRC. Enzymes in the dissolution testing of gelatin capsules. AAPS Pharm Sci Technol. 2014;15(6):1410–16.

• USP Stimuli to the Revision Process: Use of Enzymes in the Dissolution Testing of Gelatin Capsules and Gelatin-Coated
Tablets— Revisions to Dissolution and Disintegration and Dissolution of Dietary Supplements;
dx.doi.org/10.14227/DT210414P6

• Summary Report from the USP Workshop on Dissolution Testing of Capsules; dx.doi.org/10.14227/DT210414P67

• X.Song, Y.Cui, M.Xie, "Gelatin Capsule Shell Cross Linking," Pharmaceutical Technology 35 (5) 62–68 (2011)
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The contents of this post are for informational purposes only. Any opinions expressed
are solely my own and do not express the views or opinions of my employer.