Pepsin as a Case Study

for Method and Unit

Harmonization
Industry Perspective
USP Enzyme Workshop
July 8 - 9, 2009
The Spalding Auditorium
USP Headquarters, Rockville, Maryland

Presented by
Thomas K. Langdon
Vice President – Quality Assurance
American Laboratories, Inc.
Omaha, NE
` NF: “..a substance containing a proteolytic enzyme
obtained from the glandular layer of the fresh stomach
of the hog… digests not less than 3000 and not more
than 3500 times its weight of coagulated egg albumen.”
` FCC: “Obtained from the glandular layer of hog
stomach…white to light tan, water-soluble powder…”
` USP: “…prepared from the gastric mucosa of the
domestic hog (Sus scrofa L.); animals used are suitable
for human consumption. It contains gastric proteases
active in acid medium (pH 1 to 5)”.
` FIP: “..prepared from the gastric mucosa of pigs,
cattle, or sheep. It contains gastric proteases,
active in acid medium (pH 1 to 5)”.
` Ph. Eur.: “prepared from the gastric mucosa of
pigs, cattle, or sheep. It contains gastric proteases,
active in acid medium (pH 1 to 5)”.
` JP: “…a mixture of pepsin obtained from the
gastric mucosa of hog or cattle and Lactose
Hydrate…”.
` JECFA: “Commercial preparations of Pepsin
contain proteolytic enzymes obtained from the
glandular layer of the hog stomach”.
No- by all definitions pepsin is derived
from animal tissues.
` Discovered in 1836 by Theodor Schwann
` Named from the Greek word “pepsis”
(digestion)
` First animal enzyme discovered
` One of the first animal enzymes crystallized,
in 1929 by John H. Northrup.
` Pepsin Syrup Company started in 1880’s
` Beeman’s Pepsin gum – 1898
` 2009 American Laboratories, Inc. – the only
producer of pepsin in North America
` Dietary Supplement
` Digestive Aid
` Protein Hydrolysis
` Cheese making
` Leather bating (tanning process)
` Silver recovery from film (digests gelatin)
` Trichina testing (pork, horses, walrus)
` Dissolution testing
` Other?
` National Formulary – Twelfth
Edition (N.F. XII), 1965
• “Pepsin”
• “Pepsin, when assayed as herein directed digests
not less than 3000 and not more than 3500 times
its weight of coagulated egg albumen.”
w Egg albumen substrate
w Potency is determined by measurement of undigested
albumen
w Identification- “A solution of Pepsin precipitates with
solutions of tannic acid or gallic acid…”
w “Preserve Pepsin in tight containers and avoid excessive
heat.”
` United States Pharmacopeia, USP 32, 2009
• Reagents/”Pepsin, Purified”
• 1.0–1.17 Pepsin units/mg
• Hemoglobin substrate
• Spectrophotometric assay
• No identification method specified
• No storage requirements
`Food Chemicals Codex, FCC Sixth
Edition, 2008
• “Enzyme Preparations, Animal-Derived,
Pepsin”
• FCC units/mg (“One pepsin unit is defined as that quantity
of enzyme that digests 3000 times its weight of coagulated egg
albumen under the conditions of the assay.”)
• Egg albumen substrate
• Potency is determined by measurement of
undigested albumen
• No identification method specified
• “Store in tight containers in a cool, dry
place.”
` Japanese Pharmacopeia, JP XV, 2005
• “Saccharated Pepsin”
• Not less than 3800 and not more than 6000 units/g
• Casein substrate
• Spectrophotometric assay
• No identification method specified
• “Containers- Tight containers. Storage- Not
exceeding 30oC.”
` Joint FAO/WHO Expert Committee on Food
Additives (15th JECFA 1971)
• “Pepsin from Hog Stomach”
• FCC units/mg (“One pepsin unit is defined as that quantity of
enzyme that digests 3000 times its weight of coagulated egg
albumen under the conditions of the assay.”)
• Egg albumen substrate
• Potency is determined by measurement of
undigested albumen
• Identification: “…shows proteolytic activity”
• No storage requirements
` International Pharmaceutical Federation (FIP)
• Pepsin Powder (Eur. Pharm; F.I.P.)
• Not less than 0.5 pH. Eur. U. per milligram, dried
basis
• Hemoglobin substrate
• Spectrophotometric assay
• No identification method specified
• No storage requirements

Ref: Pharmaceutical enzymes, By Albert Lauwers, Simon Scharpé, Volume

84, 1997
` European Pharmacopeia 6.0, 2008
` Pepsin Powder (Pepsini pulvis)
• Not less than 0.5 Ph. Eur. U./mg dried basis
• Hemoglobin substrate
• Spectrophotometric assay
• Identification- Fibrin blue
• Store in an airtight container, protected from light,
at a temperature of 2oC to 8oC.
` USP, Pharmacopeial Forum Vol 30(1)
[Jan.-Feb. 2004].
• “Pepsin”
• Not less than 0.5 USP Pepsin Units per mg, dried basis.
• Hemoglobin substrate
• Spectrophotometric assay, “One USP Unit of Pepsin activity is the
activity that releases the equivalent of 1 μmole of tyrosine per minute
under conditions of the Assay.”
• Identification- Fibrin blue: “The Identification test is based on
the ability of Pepsin to cleave the chromophore from an insoluble
protein-dye complex.”
• “Preserve in tight containers, protected from light, and
store between 2o and 8o.”
` Milk Clot Test (Validated as Equivalent to FCC)
• Expressed as a ratio, digestive power- enzyme to
substrate (e.g. 1:10,000)
` British Pharmacopeia (BP 59)- Digestion of Egg
Albumin
` Hemoglobin Substrate- Units/mg protein
• No correlation to other methods

¾ Most methods can be adapted to other units by

experimentally determined conversion factors
e.g. 1 x USP = 0.5 USP u/mg = ~3,000 FCC
u/mg.
 FCC 3000 Equivalent Comments
Expressed as ratio, Accepted widely in US
pepsin : substrate 1:3000 and other countries
NF or NFU 3000 Archaic, still widely used
FIP or PH. Eur. 0.5 u/mg Used mostly outside of US

Neither valid nor

USP 0.5 u/mg official, usually just
“USP”, not USP u/mg.
Units/mg protein Cannot correlate to Activity largely dependent
other methods on protein content

“Pepsin” or “Pepsin No units used Illustrates the need for

Enzymes” harmonization
Assay Method Advantages
NFXI – Egg albumen Can’t think of any
digestion
FCC – Egg albumen Accepted compendial
digestion method
Milk clot test Relatively quick,
inexpensive, good accuracy
and reproducibility, can be
validated against FCC and
Ph. Eur.
Ph. Eur. And proposed USP Harmonized, good accuracy
and reproducibility.
Harmonized units with well
characterized standards
levels the playing field.
Hemoglobin assay, Units sound impressive.
expressed as u/mg protein Good for marketing.
Other units- insert YOUR With all the confusion,
OWN units here. someone will believe you.
Disadvantages
Outdated, cannot validate,
see FCC
Cumbersome, time
consuming
Somewhat subjective
Not well suited for multiple
samples, long, drawn out
test, Unnecessary steps?
Much more expensive.
Accuracy depends on 2
tests, activity and protein.
No correlation to other
methods.
You don’t really know how
potent it is. (If it’s pepsin.)
Pepsin 3000:1 45 mg Pepsin 1:3000 292 mg
Pepsin 1:10,000 units 59 mg Pepsin 220 mg
Pepsin NF 1:10,000 50 mg Pepsin Enzymes 150 mg
Pepsin 36.4 mg Pepsin NF 130 mg
Pepsin 1:10000 130 mg Pure pepsin (1:15,000) 21 mg

Pure Pepsin 1:15,000 21 mg

Pepsin 1:3000 165 mg
Pepsin USP 4 mg Pepsin (1:3,000) 175 mg
Pepsin (150 USP) 50 mg Pepsin 100 mg
Pepsin1:10 (equivalent to 14 mg
Pepsin 300NF Units 3 mg
130 mg)
Contains Pepsin NFU: 100 mg 3 mg actual pepsin or 300NF
NFU: National Formulary unit Units of activity

Pepsin (150 USP) 50 mg Pepsin (1;10,000)

NOTE: NO FCC Units Found

CONFUSED?
` NF- None specified
` FCC- None specified
` USP- Dry at 60o at 5 mm of mercury over
phosphorus pentoxide for 4 hours: it loses not
more than 5.0% of its weight.
` FIP- Not specified, although the method indicates
“with reference to the dried substance.”
` Ph. Eur.- Not more than 5.0 per cent, determined
on 0.500g by drying at 60o C over diphosphorus
pentoxide R at a pressure not exceeding 670 Pa
for 4 h.
` JP- Not more than 1.0% (1g, 80o C, 4 hours)
` JECFA- None specified
` NF- A solution of pepsin yields precipitates with
solutions of tannic acid or gallic acid and with
solutions of the salts of many heavy metals. On
heating a solution of Pepsin in acidified water to
100o, it becomes milky or yields a light,
flocculent precipitate, and loses all proteolytic
power.
` FCC- None specified
` USP- Fibrin blue
` FIP- None specified
` Ph. Eur.- Fibrin blue
` JP- None specified
` JECFA- The sample shows proteolytic activity
` How: Default method is Milk Clot Test,
validated as equivalent to FCC

` Why: Inexpensive, relatively quick, accurate

and reproducible, allows for multiple samples

` When: 3-5 days per week, every week

` What else: Ph. Eur., FCC, BP, u/mg protein

(Hgb), NF
` Where Pancreatin USP is specified to have 25 USP units/mg Protease, industry
frequently will express higher strengths as multiples of USP:
◦ 50 USP units/mg Protease = “2 x USP”
◦ 200 USP units/mg Protease = “8 x USP”, etc.
` Will this/can this be applied to Pepsin USP?
` Examples based on testing samples with different methods:
Currently, the most commonly used FCC units are 3,000, 10,000 and 15,000

“Conversion Factor”
USP u/mg X USP FCC u/mg FCC/USP
0.5 “1 x USP” 3000 6000
0.55 “1.1 x USP” 3231 5875
1.43 “2.86 x USP” 8,265 5780
1.77 “3.54 x USP” 10,390 5870
3.13 “6.26 x USP” 18,893 6038
3.27 “6.54 x USP” 20,853 6387
` Industry acceptance?
◦ Depends on how they market, if they test, how many tests they
run.
◦ Will industry accept USP units/mg or use “x USP”?
◦ Will all in industry agree that it’s always best to express the
results with appropriate units- USP u/mg?
` Possible Issues-
◦ Dietary supplement use-
x Replace all labels
x Reeducate consumers
◦ Protein hydrolysis, bulk usage-
x Completely new units means completely new calculations for protein
hydrolysis.
x Will there be any issues with equivalency after the change? Will the
same weight of pepsin in FCC units digest the same weight of protein as
pepsin in “equivalent” USP units?
x How to establish equivalency?
` Test cost, reagents, supplies, glassware
` Parts of test may be unnecessary?
◦ Double filtration
◦ Folin’s-Ciocalteu reagent
` 80% increase in labor costs. Labor is by far the
biggest cost of the test.
` 600% increase in total cost per test
` Do we adopt the method or validate a different
one?
` We still need to be proficient at the USP/pH. Eur.
Method.
` Opportunity loss due to increased labor
requirements.
 Milk-Clot Method Hemoglobin Substrate
Method

Standards $0.25 $6.00

Substrate $0.50 $3.00
Chemicals $4.01 $8.95
*Culture tubes (multi use) $1.40 $4.25
Filters $0.00 $18.00
TOTALS: $6.16 $40.20
*# Culture tubes used 28 85
` Provide for standardization of potency with either a
suitable diluent or pepsin of lower digestive power.
` Allow for higher potencies, e.g. 1.5 x USP, 3.0 x USP
etc., (where 0.5 USP units/mg = 1 x USP)
` Specify room temperature storage, pepsin is stable
without refrigeration.
` Recommend Loss on Drying testing be conducted at 4
hours under vacuum at 60oC.
` Requires a blank for each sample and standard tube,
requires each of these tubes to be double filtered
through separate prepared filter papers, filtering and
blank tubes make this test impractical/expensive/time
consuming for running multiple samples in a single run
and multiple runs in a single day
` Comment on USP and FCC monographs
` Provide stability data
` Collaborative testing
` Other method development and qualification
` Providing Pepsin reference standard
candidate material
` Providing hemoglobin substrate