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Company Confidential © 2014 Eli Lilly and Company

Linking Dissolution to Product Quality: A Case Study

Involving a Novel Cross-Linked Enzyme Therapy

Evan M. Hetrick, David C. Sperry, Hung K. Nguyen, and Mark A. Strege

Lilly Research Laboratories

Eli Lilly and Company

FDA/PQRI Conference on Evolving Product Quality

September 16th, 2014

Background: Cystic Fibrosis (CF)

• Genetic disease affecting respiratory and digestive

system

• 30,000 cases in the United States

• 70,000 cases worldwide

• Defective gene in CF patients can lead to:

• Lung infections

• Insufficient production of digestive enzymes

by the pancreas

• Treatment: pancreatic enzyme replacement therapy

(PERT)

9/19/2014 Company Confidential © 2014 Eli Lilly and Company 2

Cystic Fibrosis Foundation; http://www.cff.org

Background: PERTs

• Pancreatic Enzyme Replacement Therapy:

• Porcine-derived enzymes taken by CF patients to

improve digestion

• PERTs typically contain:

• Lipase – fats

• Amylase – carbohydrates

• Protease – protein

• While amylase and protease are important, principal

component is lipase

• Several commercially-available options exist

• Delayed-release pancrelipase


United States Pharmacopeia official monograph for Pancrelipase

Delayed-release Pancrelipase


• Pancrelipase

formulations are

typically capsules

containing enteric

coated beads to

confer delayed-

release

• Not intended to

be absorbed:

acts on

substrates in

the gut

Esophagus

Stomach

Pylorus

Duodenum

Jejunum

Ileum

Colon Small Intestine

pH 1–3 / 3–7

pH 4–6

pH 6–7

pH 7–7.5

1

2 3

Background: Liprotamase

• Three discretely produced, microbially-derived enzymes produced by

classical fermentation, purified into stable drug substances and dry-blended

with common excipients and filled into a single Size 2 capsule.

• Amylase, protease, lipase

• Classical fermentation, not porcine-derived

• Well-characterized

• Formulated to deliver specific, controlled potency over the shelf-life of the

product

• Not modified release

• Not absorbed; works on substrates in the gut


• Amylase

• Amorphous spray-dried drug substance

• Readily soluble at gastric pH

• Protease

• Crystalline drug substance

• Readily soluble at gastric pH

Lipase-CLEC

• Lipase is formulated and delivered as a cross-linked enzyme

complex (CLEC)

• Enzyme crosslinking has applications to chemical synthesis,

biosensors, pharmaceuticals, and food processing

• Performed to modulate solubility of lipase to confer stability as it

passes through the stomach


• Lipase-CLEC is essentially

insoluble at pH values less than 4.5

• Crosslinking performed with

bis(sulfosuccinimidyl) suberate

(BS3) at primary amines of lysine

residues and the N-terminus of

each polypeptide

DeSantis, G.; Jones, J.B. Curr. Opin. Biotechnol. 1999, 10, 324-330.

Margolin, A.L. Trends Biotechnol. 1996, 14, 223-230.

Lee, T.S.; Vaghjiani, J.D.; Lye, G.J.; Turner, M.K. Enzyme Microb. Technol. 2000, 26, 582-592.

Quiocho, F.A.; Richards, F.M. Proc. Natl. Acad. Sci. U.S.A. 1964, 52, 833-839.

Hetrick, E.M.; Sperry, D.C.; Nguyen, H.; Strege, M.A. Mol. Pharmaceutics 2014, 11, 1189-1200.

Lipase-CLEC

• Lipase is formulated and delivered as a cross-linked enzyme

complex (CLEC)

• Enzyme crosslinking has applications to chemical synthesis,

biosensors, pharmaceuticals, and food processing

• Performed to modulate solubility of lipase to confer stability as it

passes through the stomach


• Lipase-CLEC is essentially

insoluble at pH values less than 4.5

• Crosslinking performed with

bis(sulfosuccinimidyl) suberate

(BS3) at primary amines of lysine

residues and the N-terminus of

each polypeptide

DeSantis, G.; Jones, J.B. Curr. Opin. Biotechnol. 1999, 10, 324-330.

Margolin, A.L. Trends Biotechnol. 1996, 14, 223-230.

Lee, T.S.; Vaghjiani, J.D.; Lye, G.J.; Turner, M.K. Enzyme Microb. Technol. 2000, 26, 582-592.

Quiocho, F.A.; Richards, F.M. Proc. Natl. Acad. Sci. U.S.A. 1964, 52, 833-839.

Hetrick, E.M.; Sperry, D.C.; Nguyen, H.; Strege, M.A. Mol. Pharmaceutics 2014, 11, 1189-1200.

Enzyme Crosslinking

• For Liprotamase drug product, release of Lipase-CLEC, and other

active ingredients, is a critical quality attribute (CQA)

• Release of Lipase-CLEC is more complex than release of amylase

or protease due to intentional cross-linking and pH dependence of

solubility

• Study goal: thoroughly characterize the impact of the degree of cross-linking on the

release of Lipase-CLEC from the drug product

• Challenges:

• Generating “variably-crosslinked” Lipase-CLEC

• Methods for characterizing degree of crosslinking

• Relevant pH values for patient population

• Methods for characterizing rate of release

• Endpoint: enzyme amount vs. enzyme activity


Degree of Crosslinking (DoC)

• “Variably crosslinked” Lipase-CLEC was generated by altering the

BS3:lipase molar ratio during the crosslinking reaction


Sample BS3:Lipase Molar Ratio

A 2.0

B 3.0

C 4.0

D 6.6

Nominal cross-linking conditions (B)

• Specific enzyme activity is a

function of degree of crosslinking –

these samples remained within the

established activity specifications

(NLT 900 U/mg and NMT 3600

U/mg)

• Relevant range to study

Degree of Crosslinking


• Degree of crosslinking was

characterized by:

• Enzyme activity

• Solubility

• CE-SDS-PAGE

• Size exclusion chromatography

Incre

asin

g c

rosslin

kin

g

Lipase

monomer

standard

Linking Dissolution to Product Quality


Example CQAs

Potency

Purity

Appearance

Release

Enzyme Activity Is the enzyme still

active?

Enzyme Activity Methods


Lipase + +

Lipase generates carboxylic

acids from triglyceride

substrate

Autotitrator adds NaOH

solution to reaction to

maintain constant pH

Standard

Sample

Time

Na

OH

ad

de

d

http://us.mt..com

Enzyme Activity Analysis


• USP Apparatus 2 (single stage)

• A: pH 5.00

• B: pH 6.50

A (DoC = 2.7)

B (DoC = 3.5)

▲ C (DoC = 4.0)

D (DoC = N/A)

A (DoC = 2.7)

B (DoC = 3.5)

▲ C (DoC = 4.0)

D (DoC = N/A) In

cre

asin

g c

rosslin

kin

g

Disadvantages of Apparatus 2 / Enzyme Activity Method

• Extremely low throughput of USP lipase activity

test (2 tests / day)

• Limited dissolution timepoints for profile

• Inefficient media screen

• High variability of activity test

• To better understand product quality, a method

with the following characteristics is desirable:

• Accurate

• Precise

• Rapid


Rate of Release (Dissolution)


• Challenges: • Compendial test for delayed-release pancrelipse was not suitable for

Liprotamase

• Apparatus 1 baskets

• Enteric-coated beads vs. powder API

• Change in UV response for Lipase-CLEC

in the presence of protease

• Common protease inhibitor alters pH of

dissolution media

50

70

90

110

130

150

15 35 55 75

No

rmal

ize

d U

VR

F

Time (min)

Lipase

Lipase+Protease

5.7

5.8

5.9

6

6.1

6.2

6.3

6.4

6.5

0 20 40 60 80 100

Me

dia

pH

Time after PMSF addition

Media

pH

Time after PMSF addition

USP <711> Dissolution

http://en.wikipedia.org/wiki/File:PMSF.svg

Apparatus 4


• USP Apparatus 4 is a “flow through” system

Looney, T.J. Dissolution Technol. 1996, 3, 10-12.

Brown, W. Dissolution Technol. 2005, 12, 28-30.

Fotaki, N. Dissolution Technol. 2011, 46-49.

• Employed previously for: sustained release formulations,

suppositories, implants, powders, granules

• Primary advantage for Liprotamase: multiple dissolution media (i.e.,

various pHs) can be employed in a single run without needing to

isolate dosage unit or contents

“Open Loop”

“Closed Loop”

Apparatus 4


Principle

Exploit differential solubility of

amylase and protease vs.

Lipase-CLEC to ensure

Lipase-CLEC is not in solution

with protease.

L

Apparatus 4


P A

A P A P A A P

A P

P A A P A P A A P A P

L L

L L L

Fraction Fraction

pH

4

pH

6

Principle

Exploit differential solubility of

amylase and protease vs.

Lipase-CLEC to ensure

Lipase-CLEC is not in solution

with protease. Fraction Fraction…

Time

• Advantages:

• No proteolytic degradation

• Rapid UV endpoint

• Enables buffers with multiple pH values to

better mimic digestive tract

Dissolution profile for

Lipase-CLEC

Apparatus 4: Proof-of-Principle


AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

2.00

2.10

Minutes

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50

Fraction 1 (pH 4.00)












Protease Amylase Lipase

• After HPLC-based proof-of-principle experiments, UV endpoint was

utilized

Apparatus 4: Proof-of-Principle


Lipase-CLEC

Amylase

Protease

pH 4.0 pH 6.0

pH-dependence of Lipase-CLEC Release

• What is a relevant pH for the second stage to understand impact of

pH on Lipase-CLEC release?


Reference Duodenal pH Notes

Dressman et al., Am. J. Hosp. Pharm., 42, 2502-2506, 1985.

5-5.5 postprandial duodenal pH in CF patients

5-5.75 duodenal pH in CF patients

Geus et al., Aliment. Pharmacol. Ther., 13, 937-943, 1999.

5.25 – 6.38 (postprandial)

no pH difference btw. patients & healthy, but postprandial time <pH 5 in duodenum is significantly increased

Zentler-Munro et al., Pancreas, 7, 311-319, 1992.

<6 postprandial

Youngberg et al., Dig. Dis. Sci., 32, 472-480, 1987.

5.5-6.25 (fasting)

healthy subjects

5-6 (fasting)

CF patients

Lipase activity max at pH 8, activity drops 3-4x from pH 8 down to pH 5, irreversible inactivated below pH 4.

Gregory, J. Ped. Gastro. Nutr., 23, 513-523

≈6 (fasting) 5-7 (postprandial)

healthy subjects

≈5 (fasting) 3-6 (postprandial)

CF patients

Abrams et al., J. Clin. Invest., 73, 374-382, 1984.

5.0 basal duodenal pH in CF patients

4.9 1st

h postprandial duodenal pH in CF patients

4.4 2nd

h postprandial duodenal pH in CF patients

• pH 5.0 – 6.5 targeted for study with nominally-crosslinked Lipase-

CLEC

pH-dependence of Lipase-CLEC Release


pH 5.00

pH 5.50

pH 6.00

pH 6.25

pH 6.50

• Nominally-crosslinked Lipase-CLEC

• Stage 1: pH 4.00 for 30 min In

cre

asin

g p

H o

f S

tage 2

• Demonstrates the

impact of pH on the

dissolution of nominally-

crosslinked Lipase-

CLEC

• Potential method for

control strategy to

ensure quality

Release of Variably-Crosslinked Lipase-CLEC


A (DoC = 2.7)

B (DoC = 3.5)

C (DoC = 4.0)

D (DoC = N/A)

In

cre

asin

g D

eg

ree

of

Cro

sslin

kin

g

• Stage 2 pH 6.25

• Lot A: Lowest degree of crosslinking

• Lot D: Greatest degree of crosslinking

• SEC demonstrates presence of

monomer in Lot A

Lipase

monomer

standard

Lipase

monomer

standard

Conclusions

• Liprotamase represents a novel therapy based on

microbially-derived enzymes (vs. porcine-derived)

• Crosslinking of Lipase is a critical process parameter

that impacts the critical quality attribute of release

(dissolution)

• Novel methodologies were developed to accurately

measure Lipase-CLEC release

• Guided by an understanding of the patient population,

pH-dependence of Lipase-CLEC release was studied

• Enzymatic activity was preserved in the presence of

protease

• This strategy demonstrated the connection between

dissolution and product quality


Documents

Linking Dissolution to Product Quality: A Case Study Involving a …pqri.org/wp-content/uploads/2015/08/pdf/Hetrick.pdf · 2015-08-11 · Linking Dissolution to Product Quality: A