Human anti hemophilic factor

SUBMITTED BY-
SANTHOSH KUMAR T S
M.PHARM
1ST YEAR
PRESENTATION ON :
HUMAN ANTI HEMOPHILIC VACCINE
KARNATAKA COLLEGE OF PHARMACY
BANGALORE
FACILITATED TO-
Dr. C. SREEDHAR SIR
HEAD OF THE DEPARTMENT
(PH. ANALYSIS)

HUMAN ANTIHAEMOPHILIC FACTOR
Human anti haemophilic vaccine or Human coagulation factor VIII is a
preparation of a plasma protein fraction that contains the glycoprotein
coagulation factor VIII
It is rich in clotting factor VIII
Category:- Antihaemophilic to correct deficiencies of coagulation factor VIII
Description:- White or pale yellow powder or friable solid.
Storage:- Stored in atmosphere of nitrogen in light-resistant containers at a
temperature below 8c. The containers are sterile and sealed so as to
exclude micro- organisms.

Production:-
The plasma to be used for preparing dried human antihaemophilic fraction is
obtained from blood of healthy human donors who are, as far as can be
ascertained after clinical examination, laboratory tests on their blood and
consideration of their medical history, free from detectable agents of
infection transmissible by blood transfusion.
The examinations and tests to be carried out are decided by the National
Regulatory Authority.
In particular, the blood must be tested with negative results for:-
 Evidence of syphilic infection.
 Hepatitis-B surface antigen.
 HIV antibodies by suitably sensitive methods.
 The Haemoglobin value of the donor’s blood is not less than 12.5%w/v.

Procedure:-
 The blood is withdrawn aseptically through a closed system of sterile tubing into a
sterile container in which a suitable anticoagulant solution has been placed before
sterilization, and the container is gently agitated.
 Immediately after the withdrawal is completed, the blood is cooled to 40c, if the
plasma is to be stored frozen it is separated from the cellular components by
centrifugation and frozen to -300c or below preferably within 12hrs of collection.
 Dried human antihaemophilic fraction may be prepared from human plasma so
obtained by precipitation under controlled conditions of pH, ionic strength and
temperature with organic solvents, or by freezing and thawing.
 The precipitate may be washed by extraction with suitable solvents, dissolved in a
solution of sodium citrate adjusted to a pH of 6.8-7.2, which may also contain sodium
chloride.
 The solution is sterilized by filtration through a membrane filter, distributed in sterile
containers and dried from the frozen state.
 The air is removed or replaced by oxygen-free nitrogen and the containers are sealed so
as to exclude micro-organisms.
 No antimicrobial preservative is added but an antiviral agent may be added provided
that it can be demonstrated to have no deleterious effect on the final product in the
amount present and to cause no adverse reaction in man.

SEPARATION OF ANTIHEMOPHILIC FACTOR VIII FROM
HUMAN PLASMA BY COLUMN CHROMATOGRAPHY
 Mr.V.M.Baikar et al, has described the Column Chromatographic method for
Separation of Antihaemophilic factor VIII from Human plasma as follows:-
 TMAE-Fractogel 650 (M) (Trimethylaminoethyl) is an ion exchange medium can be
used to capture factor VIII (F VIII) directly from plasma.
 Previous reports have focused on the use of DEAE-Fractoge 1650 (M)
(Dimethylaminoethyl) ion exchange medium to capture F VIII from cryoprecipitate
and plasma.
Objectives:-
 To standardize the purification of F VIII from human plasma by column
chromatographic technique.
 To study the recovery of F VIII activity in purified fraction at 18 – 20 process
condition.
 To study the effect of virucidal step on recovery of F VIII activity
 To study the effect of lyophilisation on F VIII activity.

 In this report, Citrate Phosphate Dextrose (CPD) plasma was stirred with
dry DEAE-Sephadex A50, filtered, diluted, loaded on to a column packed
with TMAE-Fractogel and chromatographed.
 Most of the unwanted proteins flowed through the gel unadsorbed.
Bound F VIII was eluted by increasing the ionic strength of the buffer.
 This purification step gave an overall 80% recovery from the plasma with
a specific activity of 0.97 IU FVIII/mg protein.
 The purified F VIII fraction was made virus free by employing the
virucidal technique developed by New York Blood Centre (NYBC).
 There was 48.43% loss of FVIII activity in Virus inactivation treatment
and the loss of FVIII activity in lyophilisation was 8.45% which is
acceptable.
 This method of purification gave a higher yield of FVIII than
cryoprecipitation, and is a promising alternative method to
cryoprecipitation of F VIII.

Assay
Principle:
The potency of human antihaemophilic fraction is determined by comparing
the amount necessary to reduce the clotting time of a test mixture containing
substances that cause clotting of blood with the amount of standard
preparation necessary to produce the same effect under the conditions of the
following method of assay.
Standard preparation
The standard preparation is 4th international standard for blood coagulation
factor VIII:C concentrate, human, established in 1989,consisting of an
intermediate purity concentrate of human blood clotting factor VIII(supplied in
ampoules containing 6.3 units of clotting factor VIII),or another suitable
preparation the potency of which has been determined in relation to the
international standard.

Special reagents:
Normal serum reagent:
 Collect normal human blood in a dry, sterile, glass bottle; shake continuously until
coagulation is complete.
 Incubate at 37c. for 3 hours, maintain at 4c overnight,
 Remove the serum, store at -20c, Dissolve a quantity of the dried serum calculated to
have been obtained from 1 ml of the serum in sufficient imidazole buffer pH 7.4 to
produce 10 ml and allow to stand at 4 c for 16 and 24 hours
Phospholipid reagent:
 Suspend 0.125 g of Phospholipid in 5ml of water, shake and stir until a uniform
suspension is obtained.
 Prepare a dilution with saline solution that will give the concentration usually lies
between 50 and 250μg per ml.
 The diluted suspension may be kept at -20c for 6 weeks.

Clotting factor V Solution:-
 Prepare from fresh oxalated bovine plasma by fractionation at 4c with a
saturated solution of ammonium sulphate prepared at 4c.
 Use the fraction precipitating between 38% and 50% saturation, dialyzed to
remove ammonium sulphate and diluted with saline solution to give a solution
containing between 10% and 20% of the amount of clotting factor V present in
fresh normal human plasma.
Substrate plasma:-
 Separate the plasma from 9 volumes of human or bovine blood collected in 1
volume of a 3.8%w/v solution of sodium citrate .Store at -20c.
Substrate plasma deficient in clotting factor V:-
 Separate the plasma from human blood collected in one tenth its volume of a
1.34%w/v solution of sodium oxalate and incubate at 37c for 24 to 36 hours.
 Store in small amounts, at -20c.

Preparation of the test and standard solution.
Add sufficient imidazole buffer pH 7.4 to produce solutions containing
between 0.5 and 2 units per ml (stable for 15 mins at 20c).
Three successive 2-fold dilutions in the range 1 in 16 to 1 in 256 using a
mixture of 1 volume of a 3.8%w/v solution of sodium citrate and 5
volumes of saline solution as diluent.
(clotting times between 17 and 35 sec )
Introduce 0.1 ml each of clotting factor V solution, phospholipid reagent
and normal serum reagent into each of six glass incubation tubes
(75mm100mm).
To the first tube add 0.1 ml of the highest dilution of the standard
preparation place the tube in a water-bath at 37c, add 0.1 ml of 0.05M
calcium chloride and start stop-watch.

During the next minute add 0.1ml of the second highest dilution of the
standard to a second tube, place it in the water, and add 0.1 ml of 0.05M
calcium chloride at exactly 1 minute by the stopwatch.
Repeat the procedure with lowest dilution of the standard and highest to
lowest dilutions of the preparation being examined so that the calcium
chloride solution is added at 2, 3, 4 and 5 minutes by the stop-watch
,respectively
Place in a water bath at 37c twelve glass tubes each containing 0.2 ml of
0.025M calcium chloride and a further tube containing about 3 ml of
substrate plasma.
At 14 minutes, 40 seconds by the stop watch, transfer 0.1 ml of the
mixture from the first incubation tube to one of the tubes containing
0.2ml of 0.025M calcium chloride solution and mix

At 15 minutes add 0.2 ml of the warmed substrate plasma and,
using a second stop-watch, record as the clotting time the time
interval between the addition of the substrate plasma and the first
indication of fibrin formation which may be observed visually or by
mechanical means.
Repeat the procedure with the other incubation tubes at 1 minute
interval and carry out a second series of determinations at 21 to 26
minutes.
The period of incubation should, if necessary, be adjusted so that
the clotting times recorded in the corresponding tests in the two
series of determinations do not differ by more than 5%, showing
that a stable plateau of prothrombin activator formation has been
reached.
Carry out a blank determination using in place of the preparation
being examined.
An equal quantity of a mixture of 1 volume of a 3.8% w/v solution
of sodium citrate and 5 volumes of saline solution.

The result of the Assay is not valid unless the clotting time in the blank
determination is more than 40 seconds .Calculate the result of the assay
by standard statistical methods.

References:-
 Indian Pharmacopoeia- 2007;vol 3;P.no:2083-2086.
 V.M. Baikar , M.V. Kamath, et al, Separation of Antihaemophilic factor VIII
from
Human plasma; Indian Journal of Clinical Biochemistry, 2003, 18 (1) 80-86.
 http://upload.wikimedia.org/wikipedia/commons/9/9a/Protein_F8_PDB_
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