USP Disso Workshop_Tier 2 Method_JHH 11Jun2012 Final v3

Tier-2 Dissolution Method –
A Testing Lab’s Perspective
Jian-Hwa Han,
Dissolution Group, NCE-Analytical R&D,
Global Pharmaceutical Sciences, Abbott Labs
June 11, 2012 (USP Workshop – Challenges in Dissolution)

Tier-2 Dissolution, 11Jun2012
© 2012 Abbott
2
 What is gelatin cross-linking? What is the cause?
 Common practice in an Analytical/Dissolution lab
 USP <711>
Tier-2 Method
Practice
 Do we have good control of enzyme activities in the
media?
 The pepsin and pancreatin activity comparison
 Pass / Fail – Biorelevant?
Enzyme Power
 Understand the cross-linking behavior and identify the
sources of variability of Tier-2 testing
 2-step Tier-2 approach (for poorly soluble drug using
SDS as media)
Applications,
Limitations
& Remedy
Agenda

© 2012 Abbott
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Common Causes of Cross-linking
Aldehyde
• Active Ingredient
• Excipients
• Packaging Materials
• Process Environments
Temperature
Humidity
UV Light

© 2012 Abbott
4
Cross-linking? How Do I Know? – Observe Capsule Opening
(Data generated by Fiber Optic Dissolution System)
1st. - Delayed Capsule
Opening!
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60
Time, Minutes
%LC
Control 1hr-1 1hr-2 1hr-3 2hr-1 2hr-2
2hr-3 4hr-1 4hr-2 4hr-3
0
5
10
15
20
0 2 4 6 8 10
Time, Minutes
%LC
Control 1hr-1 1hr-2 1hr-3 2hr-1 2hr-2
2hr-3 4hr-1 4hr-2 4hr-3
For Example:
Fresh capsules = <2 min.;
Aged capsule = > 5 min.

© 2012 Abbott
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Visual Evidence of Cross-linking - Pellicle Formation

© 2012 Abbott
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Evidence of Cross-linking - Threading
Thin/fine tread-like materials stretching out from the
capsule shell
Need careful observation to identify

© 2012 Abbott
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Common Practice
?

© 2012 Abbott
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Use of Enzymes per USP <711>
Water or a medium with a pH of less than 6.8:
• Add purified pepsin that results in an activity
of 750,000 units/L or less
Media with a pH of 6.8 or greater:
• Add pancreatin at NMT 1750 USP units of
protease activity per Liter
Question: Do these two conditions provide
equal power to open the cross-linked gelatin
capsules?
[Ref: Jean Gallery, Jian-Hwa Han, and Chiramel Abraham, “Pepsin and
Pancreatin performance in the dissolution of cross-linked gelatin capsules
from pH 1 to 8”, Pharmaceutical Forum, 2004, Vol. 30(3), p.1084]

© 2012 Abbott
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Pepsin and Pancreatin Activity as a Function of pH
Ref: D.W. Piper and B.H. Fenton, “pH Stability
and Activity Curves of Pepsin with Special
Reference to Their Clinical Importance”, Gut
(6): 506-508, 1965
Pancreatin contains many
enzymes, including trypsin,
amylase, lipase, and
protease.
Enzyme pH Optimum
Lipase 8.0
Trypsin 7.8 ~ 8.7
Amylase 6.7 - 7.0
Pepsin
pH 5~6.8

© 2012 Abbott
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Do we have good control for enzyme activities in the
media?
Do we perform pepsin/pancreatin activity test each
time we do Tier-2 testing?
Could we rely on the vendor provided activity value
from C of A?
How much do we know about the reported activity
value?
• There are multiple testing methods for pepsin activity.
• There are many different ways to present pepsin activity
level.
Example: Pepsin, Sigma, P7000

© 2012 Abbott
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Experimental Design -
Select the right sample –
Highly soluble throughout the physiological pH range
Generate artificially cross-linked samples –
Aldehyde treatment (Daily preparation)
Evaluate Pass/Fail criteria
Observe variability and reproducibility of cross-linking
Compare method performance: Pepsin vs. Pancreatin

© 2012 Abbott
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0
20
40
60
80
100
0 10 20 30 40 50 60
Time, Minutes
%LC
Control 1-hour 2-hour 4-hour
Verification of Tier-2 Testing Results
Tier-1
Fail
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50 60
Time, Minutes
%LC
Control 1-hour 2-hour 4-hour
Pass
Similar Profiles
Tier-2
Two levels of comparison: (i) Pass the Q Spec; and (ii) Full recovery
of the profile to check the effectiveness of the enzyme digestion.
(Samples are 1, 2,
and 4 hours
treated with
formaldehyde for
both Tier-1 and
Tier-2 testing)

© 2012 Abbott
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Efficiency of Pepsin on Cross-linked Samples

© 2012 Abbott
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Efficiency of Pancreatin on Cross-linked Samples
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Time (min.)
%Released
pH 4, Tier2
pH 4, Tier1
79.2
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Time (min.)
%Released
pH 6, Tier2
pH 6, Tier1
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Time (min.)
%Released
pH6.8,Tier2
pH6.8,Tier1
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Time (min.)
%Released
pH8,Tier2
pH8,Tier1

© 2012 Abbott
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Test Comparison: 0.1N HCl/Pepsin vs pH 6.8/Pancreatin
(with 4-hour Aldehyde Treated Samples)
Tier-2, pH 1
Tier-1, pH 1
Tier-2, pH 6.8
Tier-1, pH 6.8
Control /Ref.

© 2012 Abbott
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One Step Further:
Impact of Multiple Levels of Pancreatin on Cross-linking
2 X
3 X
5 X
1X

© 2012 Abbott
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Impact on Bioequivalence - Pass/Fail Determination
 Pepsin and Pancreatin Levels were determined by the Gelatin Capsule
Working Group in 1998
 FDA BE Criteria:
80~125%
 F/P Passed;
F/F Failed Cmax –
Too High! (???)
References:
Gelatin Capsule Working Group, “Collaborative Development of Two-Tier Dissolution Testing
for Gelatin Capsules and Gelatin-Coated Tablets using Enzyme Containing Media”,
Pharmaceutical Forum 24: 7046-7050 (1998)
Marvin C. Meyer, etc., “The Effect of Gelatin Cross-Linking on the Bioequivalence of Hard
and Soft Gelatin Acetaminophen Capsules”, Pharmaceutical Research, Vol. 17, No. 8, p.
962-966 (2000)
Capsule
did open!

© 2012 Abbott
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Bioequivalence Consideration for Pass/Fail Control
 It is believed that the mildly cross-linked capsules will still be
efficacious
 Even though cross-linking can occur on stability, it is rarely
so extreme as to adversely affect performance for a well-
designed formulation
 Nevertheless, a QC measurement to evaluate the impact of
cross-linking is necessary
 Overall, 0.1N HCl with Pepsin method provides the optimal
power to digest cross-linked gelatin

© 2012 Abbott
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General Observations for Gelatin Cross-linking
 Difficult to predict when or which gelatin capsule sample will
show cross-linking
 Highly variable capsule-to-capsule behavior
 May be seen around 6 months under stressed condition
(i.e. 40°C/75RH)
 If cross-linking is observed in 6 months under stressed
condition (e.g. 40°C/75RH), similar result is expected at ~12
months under 25°C/60RH
 With a well designed Tier-2 method, the product could pass
24 months stability (Note - The objective of a Tier-2 method
is not to make the product pass – it is to eliminate the
potential artifact of cross-linking)

© 2012 Abbott
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Some Soft Spots in Current Practice

© 2012 Abbott
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Recommendations
 Identify alternative enzyme for pH ~4.5?
 2-Step Tier-2 Method for Poorly Soluble Drugs using
Surfactant Media
• Step 1: Run samples in enzyme media w/o Surfactant
• Step 2: Add surfactant concentrate to match original Tier-1 method
 2-Step Method Development Considerations:
• Volume consideration (pH, hydrodynamics)
• Media Preparation
• Step 1 treatment time (10~15 minutes)
• Verification – to make sure the method serves the purpose

© 2012 Abbott
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2-step Tier-2 Verification #1: Turbulence Impact (Avoiding
Artificial Capsule Eruption during the Addition of Medium)
 Follow 2-step Tier-2
procedures without
using enzyme for step-1
to evaluate the
turbulence impact
 Sample is treated for 4-
hours with formaldehyde
 Result: The capsules
won’t open despite the
introduced turbulence
(i.e. addition of the 2nd
solution)
2-step w/o Enzyme
Control / Tier-1
X-linked / Tier-1

© 2012 Abbott
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2-step Tier-2 Verification #2: Enzyme Power
X-linked / Tier-1
Control / Tier-2 X-linked / Tier-2
 Compare the
dissolution behaviors
of Control (untreated)
and cross-linked (4-hr
treated) samples
following the 2-step
Tier-2 procedures
 Result: The Tier-2
dissolution outcome
of cross-linked
samples matches up
with the control
samples.
Control / Tier-1

© 2012 Abbott
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Summary and Discussion
 Cross-linking problems can be difficult to overcome for
analytical labs, but they can be circumvented with a basic
knowledge of formulation and process
 It is desirable to clearly identify the enzyme activities
required for each enzyme that can provide “equivalent”
digestion power cross-linked gelatin capsules
 If the capsule opening is the only concern for cross-linked
capsule samples, it would be nice to utilize other testing
method which is simpler and better controlled.

© 2012 Abbott
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Acknowledge
 Abbott Analytical Scientists:
• Susan George
• Anagha Vaidya
• Ameesha Patel
• Daniel Bonilla
• Jean Gallery
• Chiramel Abraham
• Leonard Ferro
• Whitney Harman
• Steven Anderson
• Gregory Webster
 Abbott Management Support
for Dissolution Research and
Development
 External Inputs:
• Thomas Langdon, American
Laboratories, Inc.
• Steven Leinbach, Capsugel

© 2012 Abbott
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Questions?

© 2012 Abbott
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© 2012 Abbott
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Pepsin
 <711>: 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.
 Gastric Fluid, Simulated, TS—Dissolve 2.0 g of sodium
chloride and 3.2 g of purified pepsin, that is derived from
porcine stomach mucosa, with an activity of 800 to 2500
units per mg of protein, in 7.0 mL of hydrochloric acid and
sufficient water to make 1000 mL.
 Example: Pancreatin from porcine pancreas

© 2012 Abbott
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Pepsin (cont.)
For Example: Sigma, P7000
Before 2011: Pepsin, from porcine gastric mucosa,
Powder , 800 ~ 2500 units/mg protein
After 2011: Pepsin, from porcine gastric mucosa,
Powder , > 250 units/mg solid
USP <711> Dissolution: Pepsin, 750,000 Units (or Less) / 1000 mL
Lot # %Protein Units/mg Protein Units/ mg Solid Pepsin needed / L.
SGF,
Units/L
<711>
vs. SGF
#010M1513 49 806 1899 mg (~1.9 g) 1,263,808 59%
#050M1304V 49 920 1664 mg (~1.7 g) 1,442,560 52%
#061M1205V 376 1995 mg (~2.0 g) 1,203,200 62%
#071M0103V 473 1586 mg (~1.6 g) 1,513,600 50%

© 2012 Abbott
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Pancreatin:
 <711>: 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.
 Intestinal Fluid, Simulated, TS—Dissolve 6.8 g of
monobasic potassium phosphate in 250 mL of water, mix,
and add 77 mL of 0.2 N sodium hydroxide and 500 mL of
water. Add 10.0 g of pancreatin, mix, and adjust the
resulting solution with either 0.2 N sodium hydroxide or 0.2
N hydrochloric acid to a pH of 6.8 ± 0.1. Dilute with water to
1000 mL.
 Example: Pancreatin from porcine pancreas, 8 × USP
specifications* (Sigma, P/N. P7545; *C of A: Conforms)
 Pancreatin, 8 × USP Protease 8.0 - 9.0 x USP (American
Laboratories, Inc. specification)

© 2012 Abbott
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Pancreatin (cont.)
USP <711> Dissolution:
Pancreatin, 1,750 Units (or Less) / 1000 mL
Protease
x USP
Protease
USP
units/mg
Wt per
1000 ml
(mg)
Concentra
tion USP
units/L
Conc X
1750
8.0 200.0 8.75 1750 1.00
8.1 202.5 8.75 1772 1.01
8.2 205.0 8.75 1794 1.03
8.3 207.5 8.75 1816 1.04
8.4 210.0 8.75 1838 1.05
8.5 212.5 8.75 1859 1.06
8.6 215.0 8.75 1881 1.08
8.7 217.5 8.75 1903 1.09
8.8 220.0 8.75 1925 1.10
8.9 222.5 8.75 1947 1.11
9.0 225.0 8.75 1969 1.13
Intestinal Fluid, Simulated, TS:
Protease
x USP
Protease
USP
units/mg
Wt per
1000 ml
(mg)
Concentra
tion USP
units/L
Conc X
1750 vs.
<711>
1.0 25 10000.0 250000 143
2.0 50 10000.0 500000 286
3.0 75 10000.0 750000 429
4.0 100 10000.0 1000000 571
5.0 125 10000.0 1250000 714
6.0 150 10000.0 1500000 857
7.0 175 10000.0 1750000 1000
8.0 200 10000.0 2000000 1143
9.0 225 10000.0 2250000 1286
10.0 250 10000.0 2500000 1429
11.0 275 10000.0 2750000 1571
12.0 300 10000.0 3000000 1714
(Data Source: Tom Langdon, American Laboratories, Inc.)

© 2012 Abbott
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Reproducibility of Artificial Crosslinking Procedure:
Tier-1 Testing with 4-Hour Aldehyde Exposure
(pH Dependent!)
pH 1
pH 8

© 2012 Abbott
35
Impact of Media pH on Capsules Opening
 Capsule opening time is pH
dependent due to gelatin
solubility
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50 60 70 80
Time, Minutes
%LC
pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 6.5 pH 6.8 (n=5)
Tier-1 Testing w/o Enzymes:
Normal Capsules:
Cross-linked Capsules:
(Data Source: Steven Leinbach, Capsugel)
pH 1
pH 4
pH 4.0
pH 6.0 ~ 6.8

Tier-2 Dissolution, 21Oct2011
© 2011 Abbott
36
Delay of Capsule Opening: Tier-1 (0.1N HCl) with Varying
Degree of Cross-linking
Significant delay of capsule
shell opening for all conditions.

© 2011 Abbott
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Variability from Samples & Testing (0.1N HCl)
Sample variability (Tier-1) Testing variability (Tier-2)
4-hour Treated samples

© 2011 Abbott
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2-step Tier-2 Trial Method – Case Study
 Tier-1: [USP App 2 @ 50 RPM; 900 mL of 50 mM
Phosphate with 0.5% SDS, pH 6.8]
 Tier-2, Step-1: [USP App 2 @ 50 RPM; 500 mL of
0.1N HCl w/pepsin (750,000 unit/L)]* for 10 ~ 15 minutes
 Tier-2, Step-2: Add 400 mL 118mM Phosphate with
1.125% SDS
 Sampling time points: 15, 30, 45, and 60 minutes
(* Considering gastric exposure)

USP Disso Workshop_Tier 2 Method_JHH 11Jun2012 Final v3

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