Chromatography equipment characterization
Herein is reported a method for determining whether a re-usable chromatography column packing, which is used at least for the second time in a purification step of a purification of a polypeptide, has reduced separation efficacy in said purification step of said purification of said polypeptide, comprising the following steps: a) identifying and determining the experimental data of an inert change of at least one physicochemical parameter of a mobile phase passing through said re-usable chromatography column packing, b) determining the parameters of a function of formula I by fitting the experimental data of the inert change of the physicochemical parameter of the at least second use, c) determining the difference between the experimental data of the inert change of the physicochemical parameter of the at least second use and the function of formula I with the parameters determined in step b), d) calculating the difference between the maximum value and the minimum value of the difference determined in step c) and normalizing said difference, e) determining reduced separation efficacy of said re-usable chromatography column packing when the absolute value of the difference calculated in step d) is more than 0.1.
1. A method for determining whether a re-useable chromatography column packing, which is used at least for the second time in a purification step of a purification of a polypeptide, has reduced separation efficacy in said purification step of said purification of said polypeptide, characterized in that said method comprises the following steps:
a) identifying and determining the experimental data of an inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing of the at least second use,
b) determining the parameters of a function of formula I by fitting the experimental data of the inert change of the physicochemical parameter of the at least second use,
c) determining the difference between the experimental data of the inert change of the physicochemical parameter of the at least second use and the function of formula I with the parameters determined in step b),
d) calculating the difference between the maximum value and the minimum value of the difference determined in step c) and normalizing said difference,
e) determining reduced separation efficacy of said re-useable chromatography column packing when the absolute value of the difference calculated in step d) is more than 0.1,
wherein the function of formula I is
yI
=
-
1
2
A
·
exp
(
-
x
t
0
)
(
exp
(
2
x
c
t
0
+
w
2
2
t
0
2
)
(
erf
(
(
x
-
x
c
w
-
w
t
0
)
2
)
+
1
)
-
exp
(
x
t
0
)
·
erf
(
x
-
x
c
2
·
w
)
)
+
y
0
formula
I
with absolute signal values determined by the solvent employed (A and y 0 ), the mean value and the standard deviation of the underlying Gaussian distribution function (x c , w), the time constant of the underlying exponential decay function (t 0 ), and
wherein the erf-function used is defined as
erf
(
x
)
=
2
π
∑
0
∞
(
-
1
)
n
x
2
n
+
1
(
2
n
+
1
)
n
!
.
2. The method according to claim 1 , characterized in that said inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing is a change effected by the change of the concentration of a substance in the mobile phase that does not interact with the re-useable column packing.
3. The method according to claim 1 , characterized in that said inert change is a change in conductivity or in adsorption at 280 nm.
4. The method according to claim 1 , characterized in that said inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing is a change of from 100% of a solution containing a denaturing agent to 100% of a solution not containing said denaturing agent, or vice versa.
5. The method according to claim 4 characterized in that said denaturing agent is selected from sodium hydroxide, guanidinium chloride, urea or organic solvent.
6. The method according to claim 1 , characterized in that said inert change is a sigmoid change.
7. The method according to claim 1 , characterized in that said inert change is a change over time.
8. A method for the chromatographic purification of a polypeptide, wherein at least one chromatography step using a re-useable chromatography column packing is contained, characterized in that said method comprises the following steps:
a) identifying and determining the experimental data of an inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing of the at least second use,
b) determining the parameters of a function of formula I by fitting the experimental data of the inert change of the physicochemical parameter of the at least second use,
c) determining the difference between the experimental data of the inert change of the physicochemical parameter of the at least second use and the function of formula I with the parameters determined in step b),
d) calculating the difference between the maximum value and the minimum value of the difference determined in step c) and normalizing said difference,
wherein the function of formula I is
yI
=
-
1
2
A
·
exp
(
-
x
t
0
)
(
exp
(
2
x
c
t
0
+
w
2
2
t
0
2
)
(
erf
(
(
x
-
x
c
w
-
w
t
)
2
)
+
1
)
-
exp
(
x
t
0
)
·
erf
(
x
-
x
c
2
·
w
)
)
+
y
0
formula
I
with absolute signal values determined by the solvent employed (A and y 0 ), the mean value and the standard deviation of the underlying Gaussian distribution function (x c , w), the time constant of the underlying exponential decay function (t 0 ), and
wherein the erf-function used is defined
erf
(
x
)
=
2
π
∑
0
∞
(
-
1
)
n
x
2
n
+
1
(
2
n
+
1
)
n
!
,
and
further using the re-useable chromatography column packing when the absolute value of the difference calculated in step d) is 0.06 or less, or
performing an additional characterization and/or assessment of the purified polypeptide when the absolute value of the difference calculated in step d) is more than 0.06 but less than 0.2, or
changing the re-useable chromatography column packing when the absolute value of the difference calculated in step d) is 0.2 or more.
9. The method according to claim 8 , characterized in that said inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing is a change effected by the change of the concentration of a substance in the mobile phase that does not interact with the re-useable column packing.
10. The method according to claim 8 , characterized in that said inert change is a change in conductivity or in adsorption at 280 nm.
11. The method according to claim 8 , characterized in that said inert change of at least one physicochemical parameter of a mobile phase passing through said re-useable chromatography column packing is a change of from 100% of a solution containing a denaturing agent to 100% of a solution not containing said denaturing agent, or vice versa.
12. The method according to claim 11 , characterized in that said denaturing agent is selected from sodium hydroxide, guanidinium chloride, urea or organic solvent.
13. The method according to claim 8 , characterized in that said inert change is a sigmoid change.
14. The method according to claim 8 , characterized in that said inert change is a change over time.