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,
pepsin : substrate
1:3000
Accepted widely in US
and other countries
NF or NFU
3000
Archaic, still widely used
FIP or PH. Eur.
0.5 u/mg
Used mostly outside of US
USP
0.5 u/mg
Neither valid nor
official, usually just
“USP”, not USP u/mg.
Units/mg protein
Cannot correlate to
other methods
Activity largely dependent
on protein content
“Pepsin” or “Pepsin
Enzymes”
No units used
Illustrates the need for
harmonization
Assay Method
Advantages
Disadvantages
NFXI – Egg albumen
digestion
Can’t think of any
Outdated, cannot validate,
see FCC
FCC – Egg albumen
digestion
Accepted compendial
method
Cumbersome, time
consuming
Milk clot test
Relatively quick,
inexpensive, good accuracy
and reproducibility, can be
validated against FCC and
Ph. Eur.
Somewhat subjective
Ph. Eur. And proposed USP
Harmonized, good accuracy
and reproducibility.
Harmonized units with well
characterized standards
levels the playing field.
Not well suited for multiple
samples, long, drawn out
test, Unnecessary steps?
Much more expensive.
Hemoglobin assay,
expressed as u/mg protein
Units sound impressive.
Good for marketing.
Accuracy depends on 2
tests, activity and protein.
No correlation to other
methods.
Other units- insert YOUR
OWN units here.
With all the confusion,
someone will believe you.
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 NF
130 mg
Pepsin
36.4 mg
Pepsin 1:10000
130 mg
Pure Pepsin 1:15,000
Pepsin USP
21 mg
4 mg
Pepsin (150 USP)
50 mg
Pepsin1:10 (equivalent to
130 mg)
14 mg
Contains Pepsin NFU:
NFU: National Formulary unit
Pepsin (150 USP)
100 mg
50 mg
Pure pepsin (1:15,000)
21 mg
Pepsin 1:3000
165 mg
Pepsin (1:3,000)
175 mg
Pepsin
100 mg
Pepsin 300NF Units
3 mg actual pepsin or 300NF
Units of activity
Pepsin (1;10,000)
NOTE: NO FCC Units Found
CONFUSED?
3 mg
`
`
`
`
`
`
`
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
0.5
X USP
“1 x USP”
FCC u/mg
“Conversion Factor”
FCC/USP
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
USP u/mg
3000
6000
`
Industry acceptance?
`
Possible Issues-
◦ 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?
◦ Dietary supplement usex 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