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 Abbott2

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


Common Causes of Cross-linking

Aldehyde• Active Ingredient• Excipients• Packaging Materials• Process Environments

TemperatureHumidityUV Light


Cross-linking? How Do I Know? – Observe Capsule Opening(Data generated by Fiber Optic Dissolution System)

1st. - Delayed Capsule Opening!

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10

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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-22hr-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.


Visual Evidence of Cross-linking - Pellicle Formation


Evidence of Cross-linking - Threading

Thin/fine tread-like materials stretching out from the capsule shell

Need careful observation to identify


Testing a Capsule Sample

Follow Tier-1 Test

Protocol

Aberrant Result?

Cross-linking?

Report Results

Lab Investigation

Process

Perform Tier-2 Disso

Method

Meet Spec?

Yes

Yes

No

Yes

No

No

anticipated clinical effect?

Not Sure

Justify the Test Results

No

Follow-Up

Common Practice

?


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]


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 8.0Trypsin 7.8 ~ 8.7Amylase 6.7 - 7.0

Pepsin

pH 5~6.8


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


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-linkingCompare method performance: Pepsin vs. Pancreatin


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0 10 20 30 40 50 60

Time, Minutes

%LC

Control 1-hour 2-hour 4-hour

Verification of Tier-2 Testing ResultsTier-1

Fail

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Time, Minutes

%LC

Control 1-hour 2-hour 4-hour

PassSimilar 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)


Efficiency of Pepsin on Cross-linked Samples


Efficiency of Pancreatin on Cross-linked Samples

0.0

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Time (min.)

% R

elea

sed

pH 4, Tier 2

pH 4, Tier 179.2

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100.0

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Time (min.)

% R

elea

sed

pH 6, Tier 2

pH 6, Tier 1

0.020.040.060.080.0100.0

0 10 20 30 40 50

Time (min.)

% R

elea

sed

pH 6.8, Tier 2pH 6.8, Tier 1

0.020.040.060.080.0100.0

0 10 20 30 40 50

Time (min.)

% R

elea

sed

pH 8, Tier 2pH 8, Tier 1


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.


One Step Further:Impact of Multiple Levels of Pancreatin on Cross-linking

2 X3 X

5 X

1X


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!


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


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)


Some Soft Spots in Current Practice

Media pH

pH 4.5pH 1~2 pH 6.8

With Surfactant?

With Surfactant?

[+ Pancreatin] per USP

[+ Pepsin] per USP

Limited by Surfactant Type

YesNoYesNo


With Surfactant?

[+ Pepsin] per USP


YesNo


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


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


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


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.


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


Questions?


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


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%


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)


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)

Concentration USP units/L

Conc X 1750

8.0 200.0 8.75 1750 1.008.1 202.5 8.75 1772 1.018.2 205.0 8.75 1794 1.038.3 207.5 8.75 1816 1.048.4 210.0 8.75 1838 1.058.5 212.5 8.75 1859 1.068.6 215.0 8.75 1881 1.088.7 217.5 8.75 1903 1.098.8 220.0 8.75 1925 1.108.9 222.5 8.75 1947 1.119.0 225.0 8.75 1969 1.13

Intestinal Fluid, Simulated, TS:

Protease x USP

Protease USP units/mg

Wt per 1000 ml (mg)

Concentration USP units/L

Conc X 1750 vs. <711>

1.0 25 10000.0 250000 1432.0 50 10000.0 500000 2863.0 75 10000.0 750000 4294.0 100 10000.0 1000000 5715.0 125 10000.0 1250000 7146.0 150 10000.0 1500000 8577.0 175 10000.0 1750000 10008.0 200 10000.0 2000000 11439.0 225 10000.0 2250000 128610.0 250 10000.0 2500000 142911.0 275 10000.0 2750000 157112.0 300 10000.0 3000000 1714

(Data Source: Tom Langdon, American Laboratories, Inc.)


Selected Soft Gelatin Capsule: No cross-linking

Tier-1 Tier-2

(Q = 80% at 30 min.)


Cross-linking Levels Evaluation – Aldehyde Treated Samples(Dissolution at pH 1 with or without Pepsin)


Reproducibility of Artificial Crosslinking Procedure:Tier-1 Testing with 4-Hour Aldehyde Exposure

(pH Dependent!)

pH 1

pH 8


Impact of Media pH on Capsules Opening

Capsule opening time is pH dependent due to gelatin solubility

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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 Abbott36

Delay of Capsule Opening: Tier-1 (0.1N HCl) with Varying Degree of Cross-linking

Significant delay of capsule shell opening for all conditions.


Variability from Samples & Testing (0.1N HCl)

Sample variability (Tier-1) Testing variability (Tier-2)

4-hour Treated samples


Testing Comparison (0.1N HCl): Tier-1 vs. Tier-2

Tier-1 Tier-2


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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