Quality Standards for Medicines, Supplements, and Food Ingredients throughout the World
PEPSIN – STANDARDIZATION
CHALLENGES
Alexey Khrenov, Ph.D.
Currently Used Assays For Pepsin
• Hen egg albumen digestion
• Milk clotting assay
• Hemoglobin digestion assay (2 variants)
– Direct OD280 readout
– Development using Folin reagent
Hen egg albumen assay
• Published in USP IX, 1916
• Current assay for pepsin activity in FCC
(Appendix V)
• Assay is based on measuring the volume of
undigested albumen
Hemoglobin Assay
• First published in 1938
• Published as a Preview monograph in PF30(1)
• Used as an assay for Purified Pepsin reagent in
USP-NF
• Based on spectrophotometric measurement of
the amount of TCA-soluble material after
hemoglobin digestion by pepsin.
Milk Clotting Assay
• Currently in FCC (Appendix V), but not for
pepsin
• Based on measuring of the time required to
increase turbidity of milk solution (either
spectrophotometrically or visually)
• Commonly used in industry
Problems with Current Assays
• Most completely dependent on reference
standard
• Antiquated techniques
• Low specificity
• Low precision
• Large volumes
• Cumbersome setup
• Generally developed for determining pepsin
activity in crude food industry preparations and
not suitable for USP-NF
Need for Pepsin Assay and Unit Modernization
• New uses of pepsin in cell and tissue therapy
products manufacturing require much higher degree
of characterization of the material
• End users need more flexibility in interchanging
pepsin from different sources
Current Goals for Pepsin Assay
• Develop a highly specific pepsin assay, with a
suitable, modern substrate
• Determine the absolute pepsin activity unit or
adopt internationally recognized units
• Develop properly characterized reference
standard with known specific activity
• Establish activity conversion between assays
Approach to the Modernization of Pepsin
Documentary Standards
•
•
•
No Pepsin monograph in USP-NF
– No pepsin pharmaceutical product is currently marketed in the US
– Use of pepsin as ancillary/process material will be covered in a
general chapter on enzymes
– FCC enzyme assay needs to be in agreement with USP-NF
Two assays are planned for USP-NF/FCC
– Hemoglobin assay
– Small molecule-based assay
Unit Changes
– New single Pepsin unit will be determined
– FCC and old egg albumen assay-based units will be eliminated
Proposed small-molecule assay
• Synthetic Peptide H-Pro-Thr-Glu-Phe-p-nitroPhe-Arg-Leu-OH (Dunn et a., (1986) Biochem. J.,
237, 899-906) is used as substrate
• Hydrolysis by pepsin produces decrease in A300,
due to peak shift
• Kinetic parameters (pig pepsin): Km=80μM,
Kcat=89s-1, ΔA300 = 1000-2000 M-1 at pH=3.1.
New assay advantages
• Product release is proportional to activity, since only
one cleavage site exists in the molecule
• Does not require precipitation, filtration etc.
• Does not use harmful chemicals
• Does not require many blanks
• May be done in small volume
Work required for the new assay
• Precision estimation
• Concentrations and time linearity ranges
estimation
• Applicability study
• Interference study
• Calibration procedure development
• Formal validation and bridging to Hemoglobin
assay
Small Molecule Assay
• Pepsin
– Pepsin preparations from 2 companies were used
• Company A pepsin was used as Standard
• Company B pepsin was used as test sample to test recovery
• Assay setup
– Samples taken and OD determined for each time point
– Curves developed by plotting OD vs. activity for each time
point
– Recovery was determined by plotting results for of Test
sample on calibration curves for different time points
Small Molecule Assay
Tim e course of changes in absorbance at 300 nm
(5, 10 and 20 μ l of Pepsin solution)
Change in absorbance
(300nm)
0.1
0.08
0.06
0.04
0.02
0
-0.02
0
5
10
15
Tim e, m in
20
25
Small molecule assay
Concentrational Linearity of the assay at different time points
10 Minutes
5 Minutes
2
2
R = 0.9677
0.08
0.06
Δ A300
Δ A300
R = 0.9985
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
-0.005
0.04
0.02
0
0
0.0005
0.001
0.0015
0.002
0
0.0025
0.0005
0.001
0.0015
15 Minutes
20 Minutes
2
R = 0.904
0.08
0.08
0.06
0.06
0.04
2
R = 0.8242
0.1
ΔΑ300
Δ A300
0.0025
Pepsin Activity
Pepsin Activity
0.1
0.002
0.04
0.02
0.02
0
0
0
0.0005
0.001
0.0015
Pepsin Activity
0.002
0.0025
0
0.0005
0.001
0.0015
Pepsin Activity
0.002
0.0025
Small molecule assay
Recovery of pepsin activity
Company B pepsin activity was determined using calibration curves made with Company A pepsin
Time point used
for the activity
calculation
Activity in experiment,
units
Activity calculated,
units
% Recovery
5 min
0.001
0.0009
90%
10 min
0.001
0.0008
80%
15 min
0.001
0.0008
80%
20 min
0.001
0.0008
90%
Hemoglobin assay
• Pepsin
– Company A used for Standard Curve (hemoglobin Std.
Curve)
– Company B pepsin used for Test Recovery
•
Assay setup
– Two standard curves used to determine potency of same
Test sample (Company B) pepsin
• Tyrosine standard curve
• Hemoglobin standard curve (made with company A pepsin)
• Both curves and the test sample were run same day.
Hemoglobin Assay
Tyrosine Standard Curve
R2 = 0.9786
Absorbance (540 nm)
0.25
0.2
0.15
0.1
0.05
0
-0.6
-0.4
-0.2 -0.05 0
0.2
0.4
-0.1
-0.15
Pepsin Units/m l
0.6
0.8
1
1.2
Hemoglobin Assay
Hem oglobin Standard Cuvre
R2 = 0.9985
0.35
Absorbance (540 nm)
0.3
0.25
0.2
0.15
0.1
0.05
0
-1
-0.5
-0.05 0
0.5
1
-0.1
Pepsin Units/m l
1.5
2
2.5
Hemoglobin assay
Recovery of pepsin activity
Company B pepsin activity was determined using calibration curves made with Company A pepsin
Calibration Curve
Activity in experiment,
units
Activity calculated,
units
% Recovery
Company A Pepsin
15.50
20.2
130%
Tyrosin
15.50
20.98
135%
Summary
• Both assays are now working in the laboratory
– Comparison between assays on-going
– Formal validation of both assays is planned
• Pepsins from different sources
– Behaves differently in these assays
– For several pepsin lots we found that the activity values on CoA
are unreliable
• The reason for the different behavior of pepsins is unknown, but
may be attributed to the use of different assays and units for
activity assignments or presence of other activities
• The direct conversion between Worthington and FIP units may
be unreliable
Next Steps
• On-going studies in the lab using both assays with a
new, well-characterized pepsin, from different source
• Additional direct comparison of assays and formal
bridging study is planned
• The evaluation of new lot of USP Pepsin RS in the
assays is planned
Acknowledgements
•
•
•
•
Michael Ambrose
Lakishia James
Jeanne Fringer
Sherry Stevens
Quality Standards for Medicines, Supplements, and Food Ingredients throughout the World