Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

1990 ◽  
Vol 64 (02) ◽  
pp. 251-255 ◽  
Author(s):  
Claudine Mazurier ◽  
Armelle Parquet-Gernez ◽  
Maurice Goudemand
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Assay Method ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulant Activity ◽  
The One ◽  
In Vitro Data

SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

Decreased Potency Of Factor VIII Concentrates

1981 ◽  
Author(s):  
C K Kasper
Keyword(s):  
Factor Viii ◽  
In Vitro Assays ◽  
Assay Method ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
The One

Plasma factor VIII recoveries after infusions of factor VIII concentrates into patients with classic hemophilia have been measured in this laboratory for 14 years. Recently, we observed a decline in the in vivo recovery of factor VIII per factor VIII unit infused. In 1980, plasma factor VIII levels were measured by a one-stage APTT-based assay before and 10 min after 150 infusions of 46 lots of 3 brands of factor VIII concentrate produced in the U.S.A. Our pooled normal plasma reference was calibrated against WHO International Standard 2 and results expressed in International factor VIII units. Observed in vivo factor VIII recovery was compared to the value expected from calculations based on the unitage stated on the label. The ratio of observed/expected recovery averaged 56% per lot for brand A, 60% per lot for brand B, and 103% per lot for brand C. In vitro assays were performed on 22 lots on 36 occasions, and the ratio of observed/labelled units average 46% per lot for brand A, 53% for brand B and 75% for brand C. The two-stage factor VIII assay method of Pool and Robinson was also used to assay plasma samples from 18 infusions, and results averaged 135% of the one-stage values for infusions of brand A, 160% for brand B, and 109% for brand C. (Brand A is assayed by the manufacturer by a two-stage method, brands B and C by one-stage methods.)Decreased clinical efficacy was observed when postinfusion plasma factor VIII levels were lower than customary. The decline in potency of brands A and B has necessitated more frequent assay of patients and use of larger amounts of concentrate, with greatly-increased expense. Investigation of the effect of different assay methods and different factor VIII standards and references on the apparent factor VIII content of concentrates has begun.

Pharmacokinetic In Vivo Comparison Using 1-Stage and Chromogenic Substrate Assays with Two Formulations of Hemofil-M

1996 ◽  
Vol 76 (06) ◽  
pp. 0950-0956 ◽  
Author(s):  
Christine Lee ◽  
Trevor Barrowcliffe ◽  
Gordon Bray ◽  
Ed Gomperts ◽  
Anthony Hubbard ◽  
...  
Keyword(s):  
Factor Viii ◽  
Pharmacokinetic Studies ◽  
Chromogenic Substrate ◽  
Single Centre ◽  
One Stage ◽  
Chromogenic Assay ◽  
The Usa ◽  
The Mean ◽  
The One

SummaryIn a study to demonstrate the safety and pharmacokinetics (half-life and recovery) of two different method M purified AHF (Hemofil-M) concentrates processed in the USA and Spain, two different methods of factor VIII assay (one-stage clotting and chromogenic) have been compared in vivo. The study was a single centre blinded, randomised, crossover study. Twelve patients with severe haemophilia A (VIII: C <2 u/dl) were divided into two subgroups of six. None had received factor VIII concentrate within 48 h preceding the study. Twenty-four pharmacokinetic studies were performed in the 12 patients. Each subgroup received two different lots of study material (US and Spanish) at a dose of 50 u/kg seven days apart. A second randomisation was nominal potency, high: 1000 u or mid: 500 u per vial. The potency label was a one-stage clotting assay using the mega I standard. A standard pharmacokinetic study was performed over 24 h and each blinded sample was analysed in duplicate by a one-stage clotting (aPTT) and a chromogenic (Chromogenix AB; CS) assay at the Royal Free and NIBSC. Pharmacokinetic modelling was performed. The mean label for Hemofil-M using the chromogenic substrate assay was 79% that using the one stage assay (Mega I standard). The recovery was 17-28% higher measured by chromogenic compared to the clotting assay. Since most clinicians use the clotting assay, potency labelling using the chromogenic assay, will overestimate predicted Hemofil-M recovery by as much as 25%.

Stability and Sterility of Sucrose-Formulated Recombinant Factor VIII (Kogenate® FS/Bayer) for Use during Continuous Infusion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4004-4004
Author(s):  
Lisa Regan ◽  
Jordan Chew ◽  
Khang Vo ◽  
Joyce Leveque ◽  
Naji Khabbaz ◽  
...  
Keyword(s):  
Factor Viii ◽  
Continuous Infusion ◽  
Infusion Pump ◽  
Coagulation Activity ◽  
Excessive Bleeding ◽  
One Stage ◽  
Chromogenic Assay ◽  
Recombinant Fviii ◽  
The One

Abstract Continuous infusion of factor VIII (FVIII) is an alternative treatment approach to bolus dosing for providing hemostatic control in patients with hemophilia A undergoing surgery. Continuous infusion maintains consistent circulating FVIII, thus avoiding wastefully high or dangerously low levels. Potential advantages of continuous infusion include improved protection from excessive bleeding, reduced factor consumption, and easier monitoring. Continuous infusion requires FVIII usage parameters that are typically outside of the tested and approved ranges for the product, including extended time in an aqueous state at room temperature and exposure to a large surface area of tubing. Here we present in vitro data on the stability and sterility of a sucrose-formulated recombinant FVIII product (Kogenate® FS/Bayer; rFVIII-FS) under conditions comparable to those that would be experienced during the continuous infusion of patients. Three lots for each of 3 vial sizes (250, 500, and 1000 IU) were tested. Twenty vials of each size were reconstituted in Water for Injection, pooled into a 50 mL volume (100, 200, or 400 IU/mL for the 250, 500, and 1000 IU vial sizes, respectively), and aseptically transferred to pump reservoirs. Two mini-pumps were evaluated (WalkMed 350 Ambulatory Infusion Pump and CADD-Legacy PLUS Ambulatory Infusion Pump Model 6500) using a flow rate of 0.6 mL/hr. Reservoir bags containing reconstituted rFVIII-FS, pumps, and tubing were incubated at 30°C and samples were withdrawn to test coagulation activity via one-stage and chromogenic assays after 0.5, 1, 2, 3, 6, 12, 24, and 48 hours. Sample remaining in the reservoir after 48 hours was tested for sterility. The reservoirs and tubing used in this study were made from polyvinyl chloride (PVC), which has a high FVIII adsorption potential. Results for all 1000 IU lots based on the recoveries obtained at the first time point demonstrated &gt;80% activity at the end of the study using both the one-stage and chromogenic assays on both pumps. Results for all 500 IU lots showed similar recoveries (≥80%) for both pumps at study end using the chromogenic assay. However, the one-stage assay gave higher recovery values for the WalkMed pump (&gt;90%) compared to the CADD pump (&gt;75%). Results for all 250 IU lots demonstrated ≥79% activity at the end of the study using both pumps and both assays. All lots passed the sterility test. These findings indicate that rFVIII-FS is stable in both the WalkMed and CADD pumps and that sterility of rFVIII-FS is maintained for at least 48 hours. This suggests that rFVIII-FS is suitable for use in continuous infusion.

scholarly journals Plasma Transfusions in Hemophilia

Blood ◽  
1952 ◽  
Vol 7 (7) ◽  
pp. 710-720 ◽  
Author(s):  
S. VAN CREVELD ◽  
M. M. P. PAULSSEN
Keyword(s):  
Two Stage ◽  
Coagulation Time ◽  
Prothrombin Activity ◽  
Lasting Effect ◽  
One Stage ◽  
The One ◽  
Rapid Appearance

Abstract Transfusions of heparinized plasma have a greater and more lasting effect on the coagulation time of hemophiliacs than transfusions of citrated plasma. Both in vitro and in vivo, heparinized plasma causes in hemophiliacs a far greater consumption of prothrombin as determined with the two-stage method than citrated plasma. In using the one-stage method no important differences in prothrombin activity are found after transfusions of heparinized and of citrated plasma respectively. This fact was thought to be connected with the more or less rapid appearance of an accelerator. Its a hemophiliac with a circulating anticoagulant, transfusions of heparinized plasma were unable to shorten the coagulation time to any important degree, nor did these transfusions cause an important decrease of serum prothrombin as determined by the two-stage method.

scholarly journals Assessing the Performance of Extended Half-Life Coagulation Factor VIII, FC Fusion Protein by Using Chromogenic and One-Stage Assays in Saudi Hemophilia A Patients

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Tarek M. Owaidah ◽  
Hazzaa A. Alzahrani ◽  
Nouf S. Al-Numair ◽  
Abdulmjeed O. Alnosair ◽  
Amelita M. Aguilos ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Time Interval ◽  
One Stage ◽  
Chromogenic Assay ◽  
Significant Difference ◽  
Mean Differences ◽  
The One

Background. The one-stage assay is the most common method to measure factor VIII activity (FVIII : C) in hemophilia A patients. The chromogenic assay is another two-stage test involving purified coagulation factors followed by factor Xa-specific chromogenic substrate. Aim. This study aimed to assess the discrepancy and correlation between the chromogenic and one-stage assays in measuring FVIII : C levels in hemophilia patients receiving Extended Half-Life Elocta® as a recombinant extended half-life coagulation factor. Methods. We performed a study comparing the measurements of FVIII : C levels by the chromogenic versus the one-stage assays at different drug levels. Data of FVIII : C levels, dosage, and the time interval from administration to measurement were retrieved from the hospital records. The correlation, mean differences, and discrepancy between the two assays were calculated. The linear regression analysis was used to predict the time interval till reaching 1% FVIII : C. Results. Fourteen patients with 56 samples were included in the study. Of them, 13 patients were receiving Elocta® as a prophylactic, while one was receiving Elocta® on demand. One-third of these samples showed a discrepancy between the chromogenic and one-stage assays. The two assays were well correlated. Mean differences were significant at the individual and the time interval level. The time since the last Elocta® injection could significantly predict FVIII : C levels (β = 0.366, P<0.001). Conclusion. Our findings suggested a significant difference between both methods; the FVIII : C levels measured by the one-stage assay were less than those estimated by the chromogenic assay. However, the measurements of FVIII levels by the two assays were well correlated but discrepant in one-third of the samples. The levels of FVIII : C reach 1% after 5.4 days since the last Elocta® administration.

scholarly journals The one‐stage assay or chromogenic assay to monitor baseline factor VIII levels and desmopressin effect in non‐severe haemophilia A: Superiority or non‐inferiority?

Haemophilia ◽  
2020 ◽  
Vol 26 (5) ◽  
pp. 916-922
Author(s):  
Lisette M. Schütte ◽  
Luca S. Hodes ◽  
Iris Moort ◽  
Sara C. M. Stoof ◽  
Frank W. G. Leebeek ◽  
...  
Keyword(s):  
Factor Viii ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
One Stage ◽  
Chromogenic Assay ◽  
The One ◽  
Baseline Factor

Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa In Vivo

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 176-183 ◽  
Author(s):  
S.W. Pipe ◽  
A.N. Eickhorst ◽  
S.H. McKinley ◽  
E.L. Saenko ◽  
R.J. Kaufman
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Time Course ◽  
Hemophilia A ◽  
Specific Activity ◽  
Metabolic Labeling ◽  
Two Stage ◽  
One Stage ◽  
Thrombin Cleavage

Abstract Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation whereARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing theARG531HIS mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531HIS was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531HISdemonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,ARG531HIS had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII WT). Partially purified FVIII WT and ARG531HISproteins were subjected to functional activation by incubation with thrombin. ARG531HIS demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531HIS missense mutation predicts a charge change to the A2 subunit, we hypothesized that theARG531HIS A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster forARG531HIS compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

In Vivo Recovery and Survival of Monoclonal-Antibody-Purified Factor VIII Concentrates

1991 ◽  
Vol 66 (06) ◽  
pp. 730-733 ◽  
Author(s):  
Carol K Kasper ◽  
Hugh C Kim ◽  
Edward D Gomperts ◽  
Kenneth J Smith ◽  
Phyllis M Salzman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
In Vitro Assays ◽  
Two Stage ◽  
Double Blind ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
In Vivo Recovery

SummaryIn response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

In Vivo Recovery of Factor VIII: A Comparison of One-Stage and Two-Stage Assay Methods

1979 ◽  
Vol 42 (04) ◽  
pp. 1230-1239 ◽  
Author(s):  
I M Nilsson ◽  
T B L Kirkwood ◽  
T W Barrowcliffe
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
In Vitro Assays ◽  
Two Stage ◽  
One Stage ◽  
Significant Difference ◽  
Assay Methods ◽  
The One ◽  
Fraction I

SummaryThe recovery and half-life of VIII: C in the plasma of severely haemophilic patients was measured by one-stage and two-stage assays after injection of two Factor VIII concentrates (Hemofil, Hyland and Fraction I-O, Kabi). Plasma volumes were measured with an Evans� Blue technique, and both concentrates and post-infusion samples were measured against the same plasma standard.There was a highly significant difference in recoveries estimated by the two assay methods. The one-stage assays gave the most consistent results, in that the average recovery was 100%, whereas the two-stage assays gave only about 80% of the value expected from in vitro assays. There was no difference in recoveries between the two concentrates.The two-stage assays gave a slightly shorter half-life than the one-stage assays, and the half-life of Hemofil was also shorter than that of Fraction I-O.

A Methodological Investigation On The One-Stage Factor VIII Assay

1981 ◽  
Author(s):  
J Over ◽  
J A van Mourik ◽  
P van den Brink-Zantingh ◽  
R Smit-Jansen
Keyword(s):  
Factor Viii ◽  
Dose Response ◽  
Red Cross ◽  
Response Curves ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
Dose Response Curves ◽  
The One ◽  
Protein Rich

Assay of Factor VIII coagulant activity (VIII: C) in Factor VIII concentrates has since long met difficulties, such as l) non-paralleility of dose-response curves of plasma standard and Factor VIII concentrate, 2) spuriously low values of VIII: C in concentrates as revealed by abnormally high in vivo recoveries after transfusion, and 3) large interlaboratory variation in assay results. In an attempt to analyze the cause of these problems several parameters of the one-stage assay system were varied systematically and their effect on the parallellity of dose-response curves and on the final VIII: C value was analyzed. Nonparallel1ity was partially corrected with a protein-rich dilution medium, and almost always completely with undiluted instead of 1:1 diluted hemophilic substrate plasma. In both conditions apparently higher VIII:C values were found.A number of assay systems used by different producers of Factor VIII concentrates were compared. The standard and, in some cases, the phospholipid reagent seemed to contribute for the largest past to the inter1aboratory variation, but also other, as yet unidentified, factors exerted some influence. These findings initiated a cooperative study by five Red Cross Blood Transfusion Services in Europe on standardization of the one-stage assay for VIII:C. This resulted in a better correspondence between these institutes (CV 13%) compared to the previous situation (CV 23%).It is concluded that 1) substrate plasma should not be diluted, especially when Factor VIII concentrate is to be tested against a plasma standard, 2) the standard should be of the same type as the testmaterial, and 3) this standard should be properly calibrated against the International Standard for Factor VIII.

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