Method of controlling a polypeptide modification reaction
The invention relates to a method of controlling a polypeptide modification reaction, in particular but not exclusively, a method of controlling the activation of human factor VII (FVII) to produce human factor VII(a) (FVII(a)). The invention also relates to polypeptides obtainable by the polypeptide modification reaction and to pharmaceutical compositions comprising said polypeptides.
1. A method of obtaining a required proportion of activated factor VII, or an analogue or derivative thereof in a purified batch of factor VII/VIIa or an analogue or derivative thereof, which comprises the steps of:
(a) measuring the initial concentration of the factor VII/VIIa, or analogue or derivative thereof;
(b) measuring the initial proportion of activated factor VII, or analogue or derivative thereof;
(c) calculating the activation reaction time of the factor VII, or analogue or derivative thereof, by correlation of the values measured in each of steps (a) and (b) with a value of required proportion of activated factor VII, or analogue or derivative thereof, the reaction time, t, being calculated in accordance with formula (I):
t
=
-
ln
(
akt
0
·
(
akt
-
1
)
akt
·
(
akt
0
-
1
)
)
k
(
T
)
·
xb
·
F
0
(
I
)
wherein “akt” refers to the required proportion of activated factor VII, or analogue or derivative thereof, “akt0” refers to the initial proportion of activated factor VII, or analogue or derivative thereof, measured in step (b), “F0” refers to the initial concentration of the factor VII, or analogue or derivative thereof, (in g/l) measured in step (a), k(T) refers to the reaction constant for the given reaction (in L/g/min) as a function of temperature, T, and xb refers to the molar fraction, calculated according to the equation in formula (III),
xb
=
10
p
H
-
7.61
1
+
10
p
H
-
7.61
(
III
)
wherein pH refers to the selected pH of the reaction;
(d) performing, at a pH of 6.0 to 8.0, the factor VII activation reaction, for the time calculated in step (c); and
(e) terminating the reaction after the reaction time calculated in step (c), thereby obtaining the required proportion of the activated factor VII, or analogue or derivative thereof, wherein the terminating comprises lowering the pH to a value below about 6.0,
wherein the activated factor VII, or analogue or derivative thereof, is wt-FVIIa, K157A-FVIIa, V158T/M298Q-FVIIa, V158D/M298Q-FVIIa, V158D/M298K-FVIIa, V158D/E296V/M298Q-FVIIa, M298Q-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, S336G-FVIIa, or K337A-FVIIa.
2. The method according to claim 1 , wherein k(T) is a polynomial or a spline.
3. The method according to claim 1 , wherein the correlation procedure described in step (c) is calculated in accordance with formula (II):
t
=
-
ln
(
akt
0
·
(
akt
-
1
)
akt
·
(
akt
0
-
1
)
)
k
·
xb
·
F
0
(
II
)
Wherein “akt”, “akt0”, “k”, “xb”, and “F0” are as defined in claim 1 and k is the reaction constant.
4. The method according to claim 3 , wherein k=0.29.
5. A method of preventing degradation of an activated factor VII, or an activated factor VII analogue or derivative, in a purified batch of factor VII/VIIa or analogue or derivative thereof, to obtain a required proportion of activated factor VII, or an analogue or derivative thereof, said method comprising the steps of:
(a) measuring the initial concentration of the factor VII/VIIa, or analogue or derivative thereof;
(b) measuring the initial proportion of activated factor VII, or analogue or derivative thereof;
(c) calculating the activation reaction time of the factor VII, or analogue or derivative thereof, by correlation of the values measured in each of steps (a) and (b) with a value of required proportion of activated factor VII, or analogue or derivative thereof, the reaction time, t, being calculated in accordance with formula (I):
t
=
-
ln
(
akt
0
·
(
akt
-
1
)
akt
·
(
akt
0
-
1
)
)
k
(
T
)
·
xb
·
F
0
(
I
)
wherein “akt” refers to the required proportion of activated factor VII, or analogue or derivative thereof, “akt0” refers to the initial proportion of activated factor VII, or analogue or derivative thereof, measured in step (b), “F0” refers to the initial concentration of the factor VII, or analogue or derivative thereof, (in g/l) measured in step (a), k(T) refers to the reaction constant for the given reaction (in L/g/min) as a function of temperature, T, and xb refers to the molar fraction, calculated according to the equation in formula (III),
xb
=
10
p
H
-
7.61
1
+
10
p
H
-
7.61
(
III
)
wherein pH refers to the selected pH of the reaction;
(d) performing, at a pH of 6.0 to 8.0, the factor VII activation reaction, for the time calculated in step (c); and
(e) terminating the reaction after the reaction time calculated in step (c), thereby obtaining the required proportion of the activated factor VII, or analogue or derivative thereof, wherein the terminating comprises lowering the pH to a value below about 6.0,
wherein the activated factor VII, or analogue or derivative thereof, is wt-FVIIa, K157A-FVIIa, V158T/M298Q-FVIIa, V158D/M298Q-FVIIa, V158D/M298K-FVIIa, V158D/E296V/M298Q-FVIIa, M298Q-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, S336G-FVIIa, or K337A-FVIIa.
6. The method according to claim 1 wherein said factor VII, or analogue or derivative thereof, is V158D/E296V/M298Q-FVII(a).
7. The method according to claim 1 wherein the required proportion is between 94 and 99%.
8. The method according to claim 1 wherein the cleavage reaction additionally comprises the addition of calcium ions.
9. The method according to claim 1 , wherein the activation reaction is performed at a pH of between 6.25 and 6.75.
10. The method according to claim 3 , wherein xb=1.
11. The method according to claim 1 wherein step (d) is performed at a constant temperature.
12. The method according to claim 11 , wherein the temperature is 5-60° C.
13. The method according to claim 9 , wherein the activation reaction is performed at a pH of between 6.45 and 6.55.