1. Background
Assessment of One-Stage Coagulation Assay for KG-PF Potency testing
Juhi Patel, Yi Gao, Si Wan Kim, Amy Tollner
Quality Control Potency Department
Bayer Healthcare, Berkeley California, 94710
The goal of this project is to evaluate KG-PF One Stage coagulation
assay by Repeatability, Precision, Linearity and Accuracy studies.
This study was also done using other fill sizes which
included 250 IU/mL, 2000 IU/mL and 3000 IU/mL
and results showed similar potency as the 1000
IU/mL. All data demonstrated this method is precise,
accurate, and satisfied linear range for quantitation.
Coagulation of Factor VIII (FVIII) is tested using activated partial
thromboplastin (APTT) time based one-stage clotting assays.
One-Stage Coagulation Process
The purpose of Accuracy testing is to measure
WHO 8th standard as a sample to determine its potency
value against the KG-PF standard curve. If the measured
value of the WHO 8th meets the international standard value
of 9.4 IU/mL, the assay is accurate and can accurately
measure our product.
Results
Summary
Yi Gao, Si Wan Kim and Amy Tollner from Bayer HealthCare. Berkeley,
California. Summer Research and Development, Quality Control Potency
Department. 2015.
Acknowledgements
Accuracy Study (Against Who 8th)
Multiple preparations were made and tested from a single
vial at different times post reconstitution. The purpose was
to test how stable the material post reconstitution.
Purpose
Repeatability Study (n=6 vials)
Precision Study Stability Post Reconstitution
Linearity Study
y = 1.1106x - 38.013
R² = 0.9924
0
200
400
600
800
1000
0 100 200 300 400 500 600 700 800 900
ObservedIU/mL
Expected IU/mL
Accuracy test method: Overall mean is 108%.
This is derived from:
%Recovery =EXPECTED (9.4 IU/mL) /OBSERVED IU/mL
1. Hamilton Diluter dilutes samples to adequate concentrations
2. Load samples onto ACL Top 500 for coagulation testing.
3. ACL Top 500 CTS Coagulation Analyzer: Three levels of dilutions
are made in duplicates for parallelism analysis.
4. System suitability: A standard curve and control should meet
parallelism system suitability range and also control value has to be
within +/- 2SD range. The control range has been determined by a
historically defined mean target values.
5. All samples will be tested using this procedure for all four studies.
Conclusion
Test Procedure
Accuracy Study:
The method accurately measured WHO 8th potency
value with percentage recovery with overall 108%
from its assigned value.
Repeatability Study:
Precision % CV (N= 6 vials) for all three setups were
under 5%. The method has satisfied precision.
Stability Post Reconstitution:
Repeat testing of the single vial demonstrated the
potency values overtime with elapsed reconstitution
time is consistent 45 minutes post reconstitution.
Linearity Study:
Linearity testing shows from 50% to 200% range has
an overall % Recovery of 101% which is an average
from each level’s % recovery ranging from 95% to
107%. This means that the method is linear from 50%
to 200% according to SOP concentrations. Overall r2 =
0.9924 correlation coefficient which demonstrates it is
linear because of its closeness to the real value of 1
(100% linear).
The purpose of Repeatability testing is to measure the
closeness of the agreement between potency results of
successive measurements of the same lot material carried
out under the unchanged conditions of measurements. In
this test method, a study was conducted using 6 control
vials set against a standard to evaluate precision, which is
shown as % CV above.
The purpose of Linearity testing is to test the linear range for
quantitation. 5 levels of concentration were tested from 50-
200% range.
Instruments and Materials
Once incubated on deck of ACL Top 500 for 3-5 minutes in 37°C, all
reagents dilute with APTT reaction and immediately following the
addition of CaCl2 by the ACL Top 500, the samples begin coagulation
activity.
Instruments:
Hamilton Diluter with Venus 2 Software
ACL Top 500 CTS Coagulation Analyzer
Reagents:
Assay Standard: STD 0110 RM2
Assay Control: CTL 2093 RM1
SHP: 15P11F8, 14P7F8, 15P13F8, 14P18F8,
15P1F8, 15P11F8
Vial number
ASSAY USING 3 SEPARATE CONTROL VIALS TO TEST PRECISION
WITH RANGING RECONSTITUTION TO DILUTION TIMES
Linear testing of 1000 IU
15P11F8 14P7F8 15P13F8
Set 1 Set 2 Set 3
7043L41 7043L41 7043L54
WHO 8th
Expected Mean
%Level IU/mL IU/mL
70% 6.58 6.85 6.85 7.02 6.91
100% 9.4 10.06 10.47 10.03 10.19
130% 12.22 13.12 13.96 13.76 13.61
WHO 8th
Expected Mean
%Level IU/mL %Recovery
70% 6.58 104% 104% 107% 105%
100% 9.4 107% 111% 107% 108%
130% 12.22 107% 114% 113% 111%
Overall % 108%
Setup
Measured
IU/mL
Measured
% Recovery
SHP SHP SHP
14P18F8 15P1F8 15P11F8
7043L41 7043L41 7043L41
BeginCTL 434.98 BeginCTL 419.97 BeginCTL 437.11
CTL-1 432.2 CTL-1 422.51 CTL-1 405.47
CTL-2 418.61 CTL-2 419.97 CTL-2 394.69
CTL-3 421.15 CTL-3 402.62 CTL-3 425.41
CTL-4 399.98 CTL-4 407.50 CTL-4 422.43
CTL-5 421.25 CTL-5 367.82 CTL-5 422.47
CTL-6 394.73 CTL-6 381.36 CTL-6 440.14
EndCTL 382.13 EndCTL 412.44 EndCTL 428.24
Mean(N=6) 415 Mean(N=6) 400 Mean(N=6) 418
SD 14.3 SD 21.7 SD 16.0
CV% 3% CV% 5% CV% 4%