use of enzymes in the dissolution testing of gelatin capsules · use of enzymes in the dissolution...
TRANSCRIPT
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
Access free of charge
– Since volume 28 (2002)
Text downloaded every two months
90-day period for public comments
Searchable by keyword
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
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
5
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
7
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
8
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
9
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
10
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
11
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
12
Cross-linking in Gelatin Capsules
13
Photos supplied by Vivian Gray (VAGRAY Consulting) and Jin-Hwa Han (Abbvie)
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
16
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
17
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
18
<711> Dissolution Acceptance Table 1
19
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
20
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)
21
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
22
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
23
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
24
Pepsin
25
Piper DW, Fenton BH. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut 6:
506-508, 1965.
Pancreatin
26
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
27
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
28
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
29
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
30
Acitretin Capsules, Dissolution Test 2 http://www.usp.org/usp-nf/official-text/revision-bulletins/acitretin-capsules
Medium containing surfactants
33
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
34
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
35
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
36
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)
37
<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
38
<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
39
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
43
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
44
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
45
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