Introduction
The gelatin capsule is a widely used dosage form both for drug products as well as dietary supplements. The capsule can be made of hard or soft gelatin and it can be filled with solids, liquids or semisolids formulations. In the presence of certain compounds such as aldehydes or when exposed to high humidity and temperature, gelatin can crosslink rendering it insoluble in aqueous solvents. The presence of crosslinking will alter the dissolution behavior of the gelatin capsules; the capsules will not open and release its contents into the dissolution medium. As consequence, the product will fail the dissolution test. This failure may not reflect a possible failure to dissolve in the body. USP general chapter <711> Dissolution allows the inclusion of enzymes in the dissolution medium where gelatin capsules experience dissolution failure. The general chapter recommends the use of pepsin or pancreatin depending on the pH of the dissolution medium. This article will discuss the issues associated with the dissolution conditions for cross-linked gelatin capsules.
Gelatin
Gelatin is a purified protein obtained by partial alkaline and/or acid hydrolysis, by enzymatic hydrolysis, or by thermal hydrolysis of collagen from animals (including fish and poultry) [1]. Gelatin derived from acidcatalyzed hydrolysis is referred to as Type A, and gelatin derived from base-catalyzed hydrolysis is referred to as Type B. The main difference between gelatins derived from these two processes is in the isoeletric point (7 – 9 for acid process and 4.7 – 5.4 for alkaline process) [2].
Gelatin is graded primarily on the strength of the gel it forms and, depending on the process used and the tissue source, noticeable differences in strength are apparent among suppliers and even between lots from the same supplier. Consequently, controlling the strength of the gel from batch to batch (measured as bloom strength) is a key to obtaining a consistently performing product. Bloom strength increases when the gelatin concentration in the gel increases, when the average molecular weight of the gelatin increases, and when the pH of the gel approaches neutrality. In addition, as bloom strength increases the cost of gelatin increases and gel dissolution rate decreases. Gelatin manufacturers commonly blend different sublots of gelatin to meet bloom requirements [2].
Gelatin Capsules
Capsules are a simple way to dispense the appropriate dose of drugs in an appropriate formulation and they are an easy-to-swallow container that effectively masks the unpleasant taste of drugs. Capsules provide a useful vehicle into which multiparticulates can be filled without risk of modifying the release characteristics associated with other processing methods such as compression of multiparticulates into tablets. Since the early 1980s, technology has been available to permit accurate dosing and sealing of liquids into capsules.
Capsules can be classified into two main types based on the physical characteristics of the shell: soft gelatin capsules or softgels, and hard gelatin capsules or hardgels. Softgels have a thicker shell and typically exhibit a higher degree of elasticity because of a greater amount of added plasticizer relative to gelatin. Hardgel shells are thinner and more rigid than softgels. Both soft and hard gelatin capsules are composed of gelatin, a plasticizer, and water. For hardgels water acts as the plasticizer, whereas softgels use high-boiling-point polyols such as glycerol and sorbitol. The ratio of gelatin to plasticizer primarily determines the rigidity, brittleness, and dissolution performance of the shell.
Capsules also can be characterized by the chemical properties of the fill material (hydrophobic-based versus hydrophilic-based fill materials) or by the physical properties of the fill material (solid, solution or suspension). Hydrophobic solutions include neat oils, combinations of miscible oils, or drugs dissolved in oil vehicles. Hydrophobic suspensions include drugs suspended in oils or in oil-wax mixtures, often referred to as semisolids. Hydrophilic solutions may be neat liquids or syrups, combinations of water-soluble liquid or syrups, or drugs dissolved in water-soluble vehicles. Hydrophilic suspensions include drugs suspended in hydrophilic vehicles such as polyethylene glycol [2,3].
Cross-linking in Gelatin
Cross-linking can be seen in gelatin capsules, especially softgels, and it can occur during storage. It results in the formation during dissolution testing of swollen, rubbery water-insoluble membranes known as pellicles that may act as a barrier to drug release. A pellicle is a thin clear membrane of cross-linked protein surrounding the fill or the capsules and preventing the fill from being released. This cross-linking involves strong chemical linkages beyond simple hydrogen and ionic bonding between gelatin chains, and the linkages formed can affect the thermal reversibility of the gelatin shell. One of the strongest and most common types of cross-linking involves the covalent bonding of the amine group of a lysine side chain of one gelatin molecule to a similar amine group on another molecule. This reaction generally is catalyzed by trace amounts of reactive aldehydes. Formaldehyde, glutaraldehyde, glyoxal, and reducing sugars are the most common catalysts. The covalent bonding produced with this type of crosslinking is, for all practical purposes, irreversible, and dissolution of the shell must involve the breaking of other bonds, e.g., by enzymemediated breaking of peptide bonds in protein chains. Another, weaker, type of cross-linking is complexation of free carboxylic acid groups on two different gelatin molecules with trivalent metal ions, such as Fe3+ and Al3+. These cations may be found in some of the dyes used as colorants or as low levels of contaminants in excipients. Higher bloom gelatin, which normally is associated with higher quality, facilitates efficient cross-linking because fewer links are needed to join greater lengths of gelatin chains.
The most common causes of cross-linking include aldehydes present in any formulation component, or degradants formed in situ during storage; high humidity; decomposition of a stabilizer in corn starch resulting in the formation of aldehyde; polyethylene glycols that may auto-oxidize to form aldehydes; UV light, especially in the presence of high heat and humidity; and heat, which can catalyze aldehyde formation [2,3].
Dissolution of Cross-linked Gelatin Capsules
A well-designed dissolution test indicates when significant batch-to-batch variations occur and is a surrogate for demonstrating manufacturing consistency or significant changes in the dissolution performance of a single batch during the product’s shelf life (e.g., gelatin cross-linking). Dissolution performance also can be used as a research tool. Dissolution tests also are used to evaluate formulation design and storage conditions. More recently, dissolution testing has been used to develop in vitro/in vivo correlation (IVIVC) that is a useful tool for setting meaningful dissolution specifications and for the evaluation of post-approval changes [2].
Dissolution testing of capsules that are affected by cross-linking can yield results that suggest an apparently slower drug-release profile. However, in vivo disintegration of cross-linked capsules in healthy volunteers was found to be rapid and to be equivalent to dissolution from fresh nonstressed capsules [4]. Bioequivalence between stressed and unstressed acetaminophen hard and soft gelatin capsules also has been established [5]. These findings have led to the acceptance of a two-tier dissolution test using enzymes [6]. However, cross-linking of gelatin before or after drying the capsules can be used for obtaining extended release of the active ingredient and formaldehyde exposure has been exploited to produce enteric hard and soft capsules [7], indicating that above a certain level of cross-linking in vivo adverse effects may occur.
Based on the fact that satisfactory dissolution results are obtained for bioavailable products by adding proteolytic enzymes to the dissolution medium, the two-tier dissolution testing was included in USP 25 [7].
The current text for the two-tier dissolution testing in USP 35 [6] is: “For hard or soft gelatin capsules and gelatin-coated tablets that do not conform to the Dissolution specification, repeat the test as follows. Where water of a 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 result in an activity of 750,000 Unites 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 Unites of protease activity per 1000 mL.”
These instructions present some challenges:
- The text “…that do not conform to the Dissolution specification..” is open for interpretation because it does not relate the dissolution failure with the presence of cross-linking in the gelatin. The user can assume that the enzymes can be used for any type of failure, even those not related to gelatin cross-linking.
- The chapter recommends the use of pepsin for water or medium with a pH of less than 6.8. The pH activity curve for pepsin shows a maximum at pH 2 and almost no protease activity at pH 5.5; 70% of the maximal protease activity is still present at pH 4.5. From pH 5 to 7.5, no protease activity is possible but pepsin is stable at this range and the restoration of the pH to 2 gives maximal protease activity. Above pH 7.5 pepsin is irreversibly inactivated [8].
- The use of purified pepsin is recommended. The specification of this enzyme is in the Reagent Specifications section of USP 35 – NF 30 [9]. Enzyme activity is dependent on substrate and conditions. Not all users are aware that this specification contains the procedure for determining the appropriate pepsin activity. Commonly, the amount of pepsin to be added to the medium is based on the information displayed on the reagent label or in the certificate of analysis, where the units used may not be comparable to those obtained following the procedure in the purified pepsin specification.
- If pepsin has an acceptable activity only up to pH around 4 and pancreatin is to be used for dissolution media with pH above 6.8, a gap exists. Possible proteolytic enzymes that could be used in this intermediate pH range are papain and bromelain.
- The text does not give any guidance for dissolution media containing surfactant. Pepsin and pancreatin are not compatible with some types of surfactants [10,11]. One possible way of avoiding adverse effects by the surfactant on the enzyme is to do a pre-treatment of the cross-linked capsules. In this step, the dissolution test is run with the medium with no surfactants but with the appropriate enzyme for a short period of time, in general not more than 15 minutes. After that the surfactant is added to the medium and the dissolution is run for the duration of the test.
To address all these issues, USP created an expert panel that is evaluating possible alternatives and modifications in the twotier dissolution testing of cross-linked gelatin capsules. A revision to the USP general chapter <711> Dissolution, together with the rational for the modifications, will be published in a future issue of Pharmacopeial Forum.
References
1. USP. Gelatin. Revision Bulletin official April 1, 2013. http://www.usp.org/usp-nf/officialtext/ revision-bulletins.
2. M. R. C. Marques, E. Cole, D. Kruep, V. Gray, D. Murachanian, W. E. Brown, G. I. Giancaspro, “Liquid-filled gelatin capsules”, Pharm. Forum 2009, 35(4), 1029 – 1041.
3. Liquid-filled Capsules – dissolution Testing and Related quality Attributes <1094>, Pharm. Forum 2012, 38(1) www.usppf.com
4. J. Brown, N. Madit, E. T. Cole, I. R. Wilding, D. Cade, “The effect of cross-linking on the in vivo disintegration of hard gelatin capsules. Pharm. Res. 1998, 15, 1026 – 1030.
5. M. C. Meyer, A. B. Straughn, R. M. Mhatre, et. al. “The effect of gelatin cross-linking on the bioequivalence of hard and soft gelatin acetaminophen capsules”, Pharm. Res. 200, 17, 962 – 966.
6. USP. USP 35 – NF 30, Dissolution <711>. Rockville, MD: US Pharmacopeial Convention; 2012, 295.
7. S. Singh, K. V. R. Rao, K. Venugopal, R. Manikandan, “Alteration in dissolution characteristics of gelatin containing formulations”, Pharm. Technol. 2002, April, 36 – 58 www.pharmtech.com
8. D. W. Piper, B. H. Fenton, “pH stability and activity curves of pepsin with special reference to their clinical importance”, Gut 1965, 6, 506 – 508.
9. USP. Pepsin, purified. Rockville, MD: US Pharmacopeial Convention; 2012, 1040.
10. M. Sugikura, S. Kato, H. Tanak, “Pharmaceutical studies on enzyme preparations. VI. Influence of surface-active agents on protease activity”, Yakuzaigaku 1964, 24(4), 318 – 324.
11. 1Q. Zuo, W. Chen, X. Wang, P. Hlavacek, “Influence of surfactants on enzyme and gelatin hydrolysis”, Zhongguo Pige 2010, 39, 11 – 14.
Author Biography
Margareth R. C. Marques, M.Sc., Ph.D., studied Pharmacy and obtained her master degree in Pharmacy at the University of Sao Paulo and has a Ph.D. in analytical chemistry by the State University of Campinas. She worked in the quality control area for both active ingredients and pharmaceutical dosage forms at several pharmaceutical companies. Currently Margareth works at the U. S. Pharmacopeia where she is involved in developing general chapters for dissolution, drug release, and quality tests for some pharmaceutical dosage forms.