Use of Enzymes in the Dissolution

Testing of Gelatin Capsules

Margareth R. C. Marques, M.Sc., Ph.D. Principal Scientific Liaison

Pharmacopeial Forum (PF)

www.usppf.com

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Revisions to USP - NF

2

Early 90s

Study noncompliance of gelatin capsule products

during in vitro dissolution tests and potential

changes in bioavailability

CDER/FDA, pharmaceutical industry trade

associations, gelatin capsule manufacturers, USP,

and academia

Protocol using differentially stressed hard and soft

gelatin capsules

(Ref 4 and 11)

3

Gelatin Capsule Working Group

<711> Dissolution (USP 38)

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Chapter harmonized with the European and

Japanese Pharmacopoeias

The blue highlighted text in the previous slide is not

harmonized (USP national text).

Any revision will be done to this USP national text.

<711> Dissolution

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6

GC <2040>

<2040> DISINTEGRATION AND DISSOLUTION OF DIETARY

SUPPLEMENTS

DISSOLUTION

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 or 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 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 NMT 1750 USP Units of protease activity per 1000 mL.

RUPTURE TEST FOR SOFT SHELL CAPSULES

Medium: Water; 500 mL.

Apparatus: Use Apparatus 2 as described in Dissolution ⟨711⟩, operating at 50 rpm.

Time: 15 min

For soft gelatin capsules that do not conform to the above rupture test [ acceptance

criteria, repeat the test with the addition of purified pepsin to the Medium that results in

an activity of 750,000 Units or less per 1000 mL.

Procedure applicable to

– Any type of gelatin capsule shell

• Soft

• Hard

– Any type of filling in the gelatin capsule

• Solid (powder, granules, tablets)

• Semisolid

• Liquid

– Gelatin-coated Tablets

Critical Points

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Obtained from the partial hydrolysis of collagen

derived from the skin, white connective tissue, and

bones of animals

Most of pharmaceutical gelatin capsules are

obtained from bovine bone, bovine hide, and

porcine skin

Gelatin

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Collagen fibers Collagen helix structure

Figures from Wikipedia

From http://chemistrykitchen.blogspot.com/2010/04/marvelous-mixtures-part-2-perfect-panna.html

Composition: gelatin, plasticizer, opacifiers, colorant,

stabilizers, and buffers in an aqueous vehicle

Hard capsules – water acts as both plasticizer and the

vehicle

Soft capsules – small polyhydroxy compounds such as

glycerol, sorbitol, and maltitol typically are used as

plasticizers. Slightly longer rupture time.

Gelatin Capsules

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Strong chemical linkages beyond simple hydrogen and ionic bonding

between gelatin chains

Covalent bonding of the amine group of a lysine side chain of one gelatin

molecule to a similar amine group on another molecule

Covalent bonding is irreversible. The dissolution of the shell involves the

breaking of other bonds, e.g., by enzyme-mediated breaking of peptide

bonds in protein chains

Reaction catalyzed by trace amounts of reactive aldehydes

Cross-linking in Gelatin Capsules

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Pellicles

– Swollen, rubbery water-insoluble membrane

– Barrier to drug release

– Thin clear membrane of cross-linked protein surrounding

the fill or the capsule and preventing the fill from being

released

– Internal or external surface of the capsule

Cross-linking in Gelatin Capsules

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Evidence:

– Visual observation (see photos next slides)

– Instrumental technique (FTIR, NMR, etc.)

– Transferring the capsules contents to a non-cross-linked

capsule

Cross-linking in Gelatin Capsules

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Cross-linking in Gelatin Capsules

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Photos supplied by Vivian Gray (VAGRAY Consulting) and Jin-Hwa Han (Abbvie)

Cross-linking in Gelatin Capsules

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Photos supplied by Jian-Hwa Han (Abbvie)

Cross-linking in Gelatin Capsules

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Photos supplied by Vivian Gray (VAGRAY Consulting)

Common causes of cross-linking: – Aldehydes that are present in the active ingredient, excipients, or

packaging materials or that may be formed in-situ during storage

– High humidity associated or not with high temperature

– Substances that facilitate a cross-linking reaction

– Substances that promote decomposition of stabilizer in corn starch

resulting in the formation of ammonia and formaldehyde

– Rayon coilers that contain an aldehyde functional group (furfural)

– Polyethylene glycol may contain peroxides and aldehydes

– UV light, especially with high heat and humidity

– Aldehyde formation promoted by elevated temperatures

Cross-linking in Gelatin Capsules

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Slower release of the drug or no release at all

If there are defects in the liquid-filled capsule seam, the capsule can

rupture at the seam even in the presence of cross-linking, resulting in an

early release of the capsule fill

Degree of cross-linking is not uniform within one capsule or among

different capsules

High variability in the dissolution results

Once the cross-linking process starts it does not stop, even when the

cause is removed

Cross-linking in Gelatin Capsules

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Several papers in the literature demonstrating that

the bioavailability of the drug is not affected by the

presence of cross-linking in the gelatin capsule

(Some examples are references 1, 4, 5, 11, and

12).

Although the in vitro dissolution rate is decreased,

the extent of absorption is not reduced.

Cross-linking in Gelatin Capsules

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<711> Dissolution Acceptance Table 1

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Stage

Number

Tested Acceptance Criteria

S1 6 Each unit is not less than Q + 5%.

S2 6

Average of 12 units (S1 + S2) is

equal to or greater than Q, and

no unit is less than

Q 15%.

S3 12

Average of 24 units (S1 + S2 +S3)

is equal to or greater than Q, not

more than 2 units are less than

Q 15%, and no unit is less than Q

25%.

What is meant by “that do not conform to the

dissolution specification”?

Acceptance Table 1 has 3 stages with a total of 24

units tested.

The test can be stopped at a stage when the

results conform to the criteria.

Acceptance Tables

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If evidence of cross-linking is observed, failure of

the results to meet S1 criteria is justification for

testing with enzymes

Procedure with enzymes:

– Repeat the three stages using the original medium

modified with the appropriate enzyme

– Testing proceeds with up to 24 units tested unless

conformance to the criteria is observed at an earlier stage

– If conformance is observed during testing with enzyme-

modified medium, the sample has conformed with the

<711> Dissolution test requirement

Non conformance (Proposal)

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Pepsin

– medium with pH < 4.0

– NMT 750000 units/L

Pancreatin

– Medium with pH ≥ 6.8

– NMT 2000 USP units of protease/L

– These ranges of enzyme activities are from the results of

a collaborative study (Reference 4).

Enzymes

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Any source, most commonly porcine gastric mucosa

Pepsin, Purified in USP Reagents, Indicators and Solutions

under Reagent Specifications

Activity determined by spectrophotometry using hemoglobin

Using enzyme with higher activity allows easier filtration and

transfer of the samples

Pepsin

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Activity from the certificate of analysis and NOT

from the label (Check the procedure used)

Qualified by the appropriate procedure

Check expiry date from supplier

Revisions

– PF 38(3) [May – June 2012]

– PF 39(6) [Nov – Dec 2013]

– Future issue of PF

– Activity expressed in Units/mg using USP Pepsin for

Assay RS

Pepsin

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Pepsin

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Piper DW, Fenton BH. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut 6:

506-508, 1965.

Pancreatin

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Berdutina, AV, Neklyudov AD, Ivankin AI, Karpo BS, Mitaleva SI. Comparison of proteolytic activities of the enzyme complex from mammalian

pancreas and pancreatin. Appl. Biochem. Microbiol. 36:363-367, 2000.

USP monograph for Pancreatin

Activity determined with Assay for Protease Activity

(Casein digestive power)

Pancreatin

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Papain

– High solubility in water

– Stable in pH 2.5 – 12

– Good activity in pH 4 – 8

– USP monograph – Assay (casein digestive power)

Bromelain

– Good activity in pH 3 – 9

– Readily soluble in water

– No USP monograph, activity determination in Reagent

Specifications section of USP - NF

Proteases for the gap pH 4.0 – 6.8

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Papain and bromelain

– No relation with in-vivo conditions

– Just proteases to break the cross-linked gelatin

– Preliminary results show good behavior in dissolution with

activities of

• Bromelain 30 GDU/L (GDU = gelatin digesting units)

• Papain 550000 U/L

Proteases for the gap pH 4.0 – 6.8

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Surfactants may denature the enzyme

Pre-treatment

– Medium containing NO surfactant (Check pH) with the

enzyme

– NMT 15 min. Included in the total time of the test

• 30 min – 15 min pre-treatment + 15 min with

surfactant

– Then addition of the surfactant solution in the appropriate

concentration considering the final volume

Medium containing Surfactants

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31

<711> Dissolution (USP 39, May 1, 2016)

Example

Medium containing Surfactants

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http://www.accessdata.fda.gov/scripts/cder/dissolution/

Acitretin Capsules, Dissolution Test 2 http://www.usp.org/usp-nf/official-text/revision-bulletins/acitretin-capsules

Medium containing surfactants

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Forced cross-linking

– Exposure to formaldehyde (references 1, 3, 7, and 11)

– Contamination of one of the excipients with a known

amount of formaldehyde (reference 4)

– Exposure to high temperature/high humidity (reference 5)

Method Development and Validation

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Selection of the enzyme based on the pH of the

dissolution medium

Method for the determination of the enzyme activity

Quality grade of the enzyme (the higher the activity

the better)

If deaeration is needed, it should be done before

adding the enzyme

Method Development and Validation

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Definition of the pre-treatment in the case of

dissolution medium containing surfactant

Instructions for the analyst – how to determine that

the failure is by cross-linking

Final version of the method should contain all the

procedures for when cross-linking occurs

Method Development and Validation

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Item 2.4.2 Significant change at accelerated condition

Note: The following physical changes can be expected to

occur at the accelerated condition and WOULD NOT BE

considered significant change that calls for intermediate

testing if there is no other significant change:

– Failure to meet acceptance criteria for dissolution for 12

units of a gelatin capsule or gel-coated tablet if failure can

be UNEQUIVOCALLY ATTRIBUTED TO CROSS-LINKING

ICH Guidance Q1E (www.ich.org)

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<1094> Capsules – Dissolution Testing and

Related Quality Attributes

– Published in PF 39(3)

– Official in the First Suppl. of USP 37 (Aug 1, 2014)

– Revision will be published in a future issue of PF

Other USP General Chapters

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<1092> The Dissolution Procedure –

Development and Validation

– Official USP 29 Second Supplement (2006)

– Revision proposed in PF 40(1) (January 2014)

– Official First Supplement of USP 38 (Aug 1, 2015)

Other USP General Chapters

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European Pharmacopoeia

Japanese Pharmacopoeia

– No procedure for cross-linking in gelatin capsules

– Evaluation during registration in these two regions is

done in a case by case approach

Other Pharmacopoeias

40

1 - Singh S, Rao KVR, Venugopal K, Manikandan R. Alteration in dissolution

characteristics of gelatin-containing formulations. Pharm. Technol. April 2002,

36 – 58.

2 - Lu, X, Xiao B, Lo L, Bolgar MS, Lloyd DK. Development of a two-step tier-2

dissolution method for blinded overencapsulated erlotinib tablets using UV fiber

optic detection. J. Pharm. Biom. Anal. 56: 23 – 29, 2011.

3 - Gallery J, Han JH, Abraham C. Pepsin and pancreatin performance in the

dissolution of crosslinked gelatin capsules from pH 1 to 8. Pharm. Forum 30(3)

[May – June 2004].

4 – Collaborative development of two-tier dissolution testing for gelatin capsules and

gelatin-coated tablets using enzyme-containing media. Pharm. Forum 25(5):

7045 – 7050, 1998.

References

41

5 – Bottom CB, Clark M, Carstensen JT. Dissolution testing of soft shell capsules –

acetaminophen and nifedipine. J. Pharm. Sci. 86(9), 1057 – 1061, 1997.

6 – Rao KVR, Singh S. Sensitivity of gelatin raw materials to cross-linking. Pharm.

Technol. December 2002, 42 – 46.

7 – Marchais H, Cayzeele G, Legendre JY, Skiba M, Arnaud P. Cross-linking of

hard gelatin carbamazepine capsules: effect of dissolution condition on in vitro

drug release. Eur. J. Pharm. Sci. 19: 129 – 132, 2003.

8 – Ofner CM, Zhang YE, Jobeck VC, Bowman BJ. Crosslinking studies in gelatin

capsules treated with formaldehyde and in capsules exposed to elevated

temperature and humidity. J. Pharm. Sci. 90(1): 79 – 88, 2001.

References

42

9 – Pennings FH, Kwee BLS, Vromans H. Influence of enzymes and surfactants

on the disintegration behavior of cross-linked hard gelatin capsules during

dissolution. Drug Dev. Ind. Pharm. 32: 33 – 37, 2006.

10 – Tengroth C, Gasslander U, Andersson FO, Jacobsson SP. Cross-linking of

gelatin capsules with formaldehyde and other aldehydes: an FTIR

spectroscopy study. Pharm. Dev. Technol. 10: 405 – 412, 2005.

11 – Meyer MC, et. al. The effect of gelatin cross-linking on the bioequivalence of

hard and soft gelatin acetaminophen capsules. Pharm. Res. 17(80): 962 – 966,

2000.

12 – Digenis GA, Gold TB, Shah VP. Cross-linking of gelatin capsules and its

relevance to their in vitro – in vivo performance. J. Pharm. Sci. 83(7): 915 –

921, 1994.

References

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13 – Digenis GA, Sandefer EP, Page RC, Doll WJ, Gold TB, Darwazeh NB.

Bioequivalence study of stressed and nonstressed hard gelatin capsules using

amoxicillin as a drug marker and gamma scintigraphy to confirm time and GI

location of in vivo capsule rupture. Pharm. Res. 17(5): 572 – 582, 2000.

14 – Kalantzi L, Page R, Nicolaides E, Digenis G, Reppas C. In vitro methods can

forecast the effects of intragastric residence on dosage form performance. Eur. J.

Pharm. Sci. 33: 445 – 451, 2008.

15 – Brown J, Madit N, Cole ET, Wilding IR, Cade D. The effect of cross-linking on

the in vivo disintegration of hard gelatin capsules. Pharm. Res. 15(7): 1026 –

1030, 1998.

16 – Zhao F, Malayev V, Rao V, Hussain M. Effect of sodium lauryl sulfate in

dissolution media on dissolution of hard gelatin capsule shells. Pharm. Res.

21(1): 144 – 148, 2004.

References

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17 – Marques MRC, Cole E, Kruep D, Gray V, Murachanian D, Brown WE,

Giancaspro GI. Liquid-filled Gelatin Capsules. Pharm. Forum 35(4) [July – aug

2009].

18 – Song X, Cui Y, Xie M. Gelatin Capsule shell cross-linking. Tier II dissolution

method development in the presence of sodium lauyrl sulfate. Pharm. Technol.

May 2011, 62 – 68.

19 – Stein D, Bindra DS. Stabilization of hard gelatin capsule shells filled with

polyethylene glycol matrices. Pharm. Dev. Technol. 12: 71 – 77, 2007.

20 – Guyot M, Fawaz F, Maury M. In vitro release of theophylline from cross-linked

gelatin capsules. Int. J. Pharm. 144: 209 – 216, 1996.

References

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www.dissolutiontech.com

www.americanpharmaceuticalreview.com

www.pharmtech.com

www.tabletscapsules.com

www.usppf.com

www.accessdata.fda.gov/scripts/cder/dissolution/index.cfm

www.usp.org

Useful websites

46

Margareth Marques, M.Sc., Ph.D.

E-mail MRM@usp.org

Phone 301 816 8106

Contact information

47