Tip: Using enzymes in dissolution testing of gelatin capsules
G. Bryan Crist, Agilent Scientific Affairs Manager
A concern identified with the existing Dissolution Chapter has been the efficiency of enzymes for two-tier testing in the event of a dissolution failure due to cross-linking. A modification to USP Chapters: Dissolution and Disintegration and Dissolution of Dietary Supplements has been published in the US Pharmacopeial Forum.  In March of 2014, at USP headquarters in Rockville, MD, a workshop was conducted to address the ineffectiveness of presently used pepsin and pancreatin in handling dissolution of gelatin capsules exhibiting crosslinking in media with a mid-range pH of about 4.5 to 6.5 units. [2,3]
Cross-linking can render dissolution testing and monitoring useless
Cross-linking is defined as the “formation of strong chemical linkages beyond simple hydrogen and ionic bonding between gelatin chains.” The reaction itself is:
Renders the gelatin insoluble
Catalyzed by a number of chemical and environmental factors 
Figure 1. Cross-linking and pellicle formation.
Figure 2. Peptic and protease activity with respect to pH.
The impact of cross-linking is a delay or inability of the capsule shell to open due to pellicle formation on the internal or external gelatin surface (Figure 1). This is visually confirmed with the presence of thin, rubbery-appearing membranes or gelatinous masses. Occasionally, thin wispy strands of gelatin may be observed, indicating early signs of cross-linking during stability studies.
The current ICH-harmonized USP requirement for hard or soft gelatin capsules and gelatin-coated tablets that do not conform to the dissolution specifications is:
Where water or medium with a pH of less than 6.8 is specified as the Medium in the individual monograph, the same Medium specified may be used with the addition of purified pepsin that results in an activity of 750,000 Units or less per 1000 mL – for media with a pH of 6.8 or greater, pancreatin can be added to produce not more than 1750 USP units of protease activity per 1000 mL.
The problem with this approach is that the activity of pepsin peaks around pH 2 and has good activity up to about pH 4.5. Pancreatin peak activity is about pH 6.8 with good activity between 6-8 units. This creates a problem for products requiring media in the range of about 4 to 6.8 because the listed enzymes do not have sufficient activity in this range, as illustrated in Figure 2.
Two new enzymes shown promising in elimination of cross-linking
The USP workshop suggested two new enzymes with sufficient activity in mid-range pH media: papain and bromelain.  These were also included in the US Pharmacopeial Forum for inclusion into the USP Dissolution chapter. Papain is derived from unripe green papaya while bromelain is derived from pineapple stems. Both have an optimal pH around 5 and exhibit good activity through the pH range of 4.0 to 6.8. The proposed activity levels are NMT 550,000 units/L for papain and NMT 30 gelatin-digesting units (GDU)/L of dissolution media for bromelain. The activity determinations are found in the USP under the monograph section for papain and under the Reagent Specifications for bromelain. Additionally, the continued use with pepsin will be clarified for use with pH ≤ 4.0.
Enzyme interaction with surfactant containing dissolution media and product formulations during the dissolution method development stages should be evaluated with “healthy” gelatin capsules. If additional testing is to be performed with enzymes due to cross-linking, the establishment of the proper enzyme for Tier 2 testing will have been verified and documented. To learn more about this topic, visit the Dissolution Discussion Group (DDG) page and listen to the May 2015 DDG quarterly meeting replay.
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