DESC:FIELD OF INVENTION
The present invention relates to an analytical method for measuring inorganic anions present in a protein sample by ultrafiltration and chromatography.
BACKGROUND OF INVENTION
A protein-based therapeutic composition must be analyzed for inactive constituents present in the composition along with the active pharmaceutical ingredient throughout the pre-formulation and formulation stages. This includes inorganic anions in a biologic drug sample – introduced into the sample either as a part of sample processing, or present as a constituent of one or more excipient of the finished drug product.
Inorganic anions in protein compositions are usually measured by employing ultrafiltration followed by ion chromatography. This can be challenging in a protein-based sample owing to interaction of the ions with the protein itself. One such phenomenon is referred to as Donnan effect. Donnan effect refers to the behavior of certain charged particles near a semi-permeable membrane, usually due to the presence of another impermeable charged particle in the vicinity, resulting in uneven distribution of ions across said membrane. Thus, in the case of a solution containing monoclonal antibodies, negatively charged small ions present in the solution can bind to the positively charged protein molecules, thereafter, not diffusing across the semi-permeable membrane during ultrafiltration, thereby altering the distribution of the nature of ions across the membrane. Consequently, the measured levels of the analyte can differ significantly from their actual levels in the protein solution. Donnan effect can interfere in the analytical measurements of both in-process (under development) and finished drug product samples as mentioned above. This is more pronounced in the case of high concentration protein formulations wherein increased electrostatic interactions between the protein and ions can create greater disparity between actual and measured levels of the analyte.
Presently, efforts to mitigate Donnan effect is attempted using prediction models in monoclonal antibody processes. However, no definitive solutions to mitigate Donnan effect in analytical determination of anions in monoclonal antibody solution can be found. For this reason, it is important to develop a reliable method that can be used across a range of monoclonal antibody preparations to account for Donnan effect in preformulation as well formulation studies.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a method for measuring inorganic anions present in a protein sample. The method employs use of a diluent comprising histidine, followed by ultrafiltration and chromatography that can mitigate Donnan effect substantially. The method when performed as disclosed, can increase the percentage recovery of the analyte by up to at least 13% when compared to a method devoid of the parameters as disclosed. The aforementioned parameters would include but are not limited to one or more of the dilution fold of the sample, constitution of diluent and centrifugation time. The method is particularly advantageous in case of detecting anions in high concentration protein solutions (as high as 125 mg/mL) and in that it can serve as a platform method that can be employed across a range of monoclonal antibody formulations.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Phosphate standard curve (1-8 µg)
Figure 2: Chloride standard curve (1-8 µg)
Figure 3: Chromatogram of Tocilizumab (intravenous; 20 mg/mL) sample showing chloride and phosphate peaks
Figure 4: Chromatogram showing chloride and phosphate peaks of subcutaneous (SC; 125 mg/mL) and intravenous (IV; 25 mg/mL) Abatacept samples
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “biologic” used herein refers to any diagnostic, preventive, or therapeutic preparation, such as serum, vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins derived from animal products or other biological source.
The term “diluent” used herein refers to any liquid substance that is mixed with a solution to reduce its viscosity and increase its flow rate.
The term “monoclonal antibody” used herein refers to laboratory produced antibodies designed to recognise and bind to specific receptors found on the cell surface, intracellular or extracellular space.
The term “suppressor” or “anion suppressor” used herein refers to a device or a part of ion chromatography system which is used to reduce background noise levels and to enhance analyte peak response during detection. The suppressor removes conductive ions from the eluent (thus reducing background and in turn increasing sensitivity), removes counter ions in the sample (e.g, removes cations where anions are of interest and vice versa) and converts the ion of interest into a more conductive form.
The term “substantially” used herein refers to the fact the method when performed as disclosed, can increase the percentage recovery of the analyte (anion species in present invention) by up to at least 13% when compared to a method devoid of the parameters as disclosed. The aforementioned parameters would include but are not limited to one or more of dilution fold of the sample, constitution of diluent and centrifugation time.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person having ordinary skill in the art to which the invention pertains.
Various embodiments of the disclosed invention provide a rapid and sensitive method for detection and quantitation of anion species present in a protein sample by employing parameters such as but not limited to one or more of dilution fold of the sample, constitution of diluent and centrifugation time, wherein the method mitigates Donnan effect substantially.
The disclosed method is particularly advantageous in case of detecting anions in high concentration protein solutions (as high as 125 mg/mL) and in that it can serve as a platform method that can be employed across a range of monoclonal antibody formulations.
In an embodiment, the invention discloses a method for measuring anion species present in a protein sample, wherein the method comprises:
a) diluting the sample in a solution containing histidine;
b) centrifuging the sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) subjecting the permeate of step b) to suppressed ion chromatography column with a mobile phase comprising sodium carbonate and sodium bicarbonate;
d) collecting the eluate containing the anion species under isocratic conditions;
e) detecting anion species in the eluate using an electrical conductivity detector; and
f) comparing the chromatographic profile of the eluate with that of a standard curve, thereby measuring anion species in the protein sample;
wherein the method mitigates Donnan effect substantially.
In a further embodiment, the concentration of histidine is 10 mM to 20 mM.
In yet another embodiment, the concentration of histidine is 16 mM.
In another embodiment the sample is diluted at least about 5 fold, at least 6 about fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least 15 fold or at least 20 fold.
In another embodiment the sample is diluted preferably at least 5 fold.
In another embodiment the sample is diluted preferably at least 10 fold.
In another embodiment the sample is diluted preferably at least 20 fold.
In yet another embodiment, the sample is centrifuged using a centrifugal filter at 2900 g for 30 minutes.
In yet another embodiment, the sample is centrifuged using a centrifugal filter at 2900 g for 40 minutes.
In another embodiment the protein is a monoclonal antibody or a fusion protein.
In yet another embodiment the anion species are one or more of phosphate or chloride ions.
In another embodiment, the mobile phase contains 4.5mM Sodium carbonate and 1.4mM Sodium bicarbonate
In yet another embodiment, the flow rate is 0.8 to 1.5 mL/min
In another embodiment the method mitigates Donnan effect by increasing recovery of anion species by up to at least 13% when compared to using a diluent devoid of the disclosed parameters.
In yet another embodiment background conductivity of anion suppressor is set from 17 – 25 µS/cm.
In an embodiment, the invention discloses a method for measuring phosphate ions present in a protein sample, wherein the method comprises:
a) diluting the sample in a solution containing histidine;
b) centrifuging the sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) subjecting the permeate of step b) to suppressed ion chromatography column with a mobile phase comprising sodium carbonate and sodium bicarbonate;
d) collecting the eluate containing the anion species under isocratic conditions;
e) detecting anion species in the eluate using an electrical conductivity detector; and
f) comparing the chromatographic profile of the eluate with that of a standard curve, thereby measuring phosphate ions in the protein sample;
wherein the method mitigates Donnan effect substantially.
In an embodiment, the invention discloses a method for measuring chloride ions present in a protein sample, wherein the method comprises:
a) diluting the sample in a solution containing histidine;
b) centrifuging the sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) subjecting the permeate of step b) to suppressed ion chromatography column with a mobile phase comprising sodium carbonate and sodium bicarbonate;
d) collecting the eluate containing the anion species under isocratic conditions;
e) detecting anion species in the eluate using an electrical conductivity detector; and
f) comparing the chromatographic profile of the eluate with that of a standard curve, thereby measuring chloride ions in the protein sample;
wherein the method mitigates Donnan effect substantially.
In an embodiment, the invention discloses a method for measuring phosphate and chloride ions present in a protein sample, wherein the method comprises:
a) diluting the sample in a solution containing histidine;
b) centrifuging the sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) subjecting the permeate of step b) to suppressed ion chromatography column with a mobile phase comprising sodium carbonate and sodium bicarbonate;
d) collecting the eluate containing the anion species under isocratic conditions;
e) detecting anion species in the eluate using an electrical conductivity detector; and
f) comparing the chromatographic profile of the eluate with that of a standard curve, thereby measuring phosphate and chloride ions in the protein sample;
wherein the method mitigates Donnan effect substantially.

In yet another embodiment, the protein sample is vedolizumab sample.
In yet another embodiment, the protein sample is tocilizumab sample.
In yet another embodiment, the protein sample is adalimumab sample.
In yet another embodiment, the protein sample is abatacept sample.
Abbreviations
API : Active Pharmaceutical Ingredient
AUC : Area Under the Curve
FB : Formulation Buffer
FDA : Food and Drug Administration
FP : Fusion Protein
IV : Intravenous
RSD : Regression standard deviation
RT : Retention Time
SC : Subcutaneous
TFFR : Tangential Flow Filtration Retentate
WFI : Water for Injection
EXAMPLES
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1: Estimation of phosphate ions in a protein sample
For estimation of phosphate ions in sample, standard curve was first generated by injecting varying amounts (1 to 8 µg) of phosphate (using the same stock of working standard) in an increasing order (increasing injection volumes) into an ion chromatography column in duplicates employing suppressor and thereafter, detecting phosphate ions using a conductivity detector. A graph is plotted with amount of phosphate (in µg) on X-axis and peak area (AUC) on the Y-axis. The typical chromatographic profile of Phosphate will have one major peak at RT ~ 4.7 ± 0.5 minutes.
From the plots, it was found that phosphate standards ranging from 1-8 µg was linear with a regression (R2) = 0.99 (Table 1, Figure 1) and % RSD within 2.1 - 3.7.
Standard amount (µg) Standard preparation % RSD
Peak area (µS/minutes)
1 1.33 2.42
2 2.97 3.70
3 4.70 2.59
4 6.63 2.46
5 8.62 2.41
6 10.69 2.22
7 12.61 2.07
8 14.18 2.08

Table 1
Next, analysis of phosphate in protein sample was performed. For this, sample was diluted 4 to 10 fold in diluent containing 16 mM Histidine in water. 1 mL of diluted protein sample was then loaded onto 30 kDa cut off centrifugal filter and spun at 2900 g for 30-40 minutes. The retentate (containing protein) was discarded and the permeate (containing analyte, which is phosphate in present case) was collected for subsequent analysis. 10-50 µL said permeate was injected into and run through a suppressed ion chromatography column at a flow rate of 0.8 – 1.5 mL/min. Mobile phase consisted of 4.5mM Sodium carbonate and 1.4mM Sodium bicarbonate. The sample was eluted from the column under isocratic conditions for a maximum run time of about 8 minutes and detected using a conductivity detector. Based on the chromatographic profile of the multiple test sample runs and comparing the same with the standard curve, average concentration of phosphate ions is calculated.
Concentration of phospate (mM) in test sample was calculated using the equation:

A representative data for result of observed phosphate ion concentration from 6 different runs of in-house tocilizumab (intravenous, 20 mg/mL) samples (4 fold diluted in 16 mM histidine) is shown in Table 2.

Samples Observed Conc. (mM) in different injections Avg conc. (mM) SD
1 2 3 4 5 6
Tocilizumab (intravenous) 3.47 3.48 3.47 3.49 3.48 3.48 3.48 0.01
Table 2
In case of validation studies, % recovery of phosphate ions was calculated using the equation:

Representative data for in-house abatacept (125 mg/mL) samples (5 or 10 fold diluted) and in-house vedolizumab (60 mg/mL) samples indicating % recovery of phosphate ions calculated as per above equation are shown in Table 3 & 4 respectively.
Sample ID Injection volume (µL) Area (µS*min) Observed phosphate Conc. (mM) Expected conc.(mM) % Recovery
Abatacept_5X dilution_Run1 25 7.76 9.6 10 96
Abatacept_5X dilution_Run2 25 7.75 9.6 10 96
Abatacept_10X dilution_Run1 50 7.33 9.1 10 91
Abatacept_10X dilution_Run2 50 7.32 9.1 10 91
Table 3
Sample ID Inj. Vol. (µL) Area (µS* min) Observed phosphate Conc. (mM) Expected conc.
(mM) %Recovery
Vedolizumab_10X dilution_Run1
50 4.25 5.85 6.9 84.6
Vedolizumab_10X dilution_Run2
50 4.26 5.84 6.9 84.8
Table 4
Example 2: Estimation of chloride ions in a protein sample
For estimation of chloride ions in sample, standard curve was first generated by injecting varying amounts (1 to 8 µg) of chloride (using the same stock of working standard) in an increasing order (increasing injection volumes) into an ion chromatography column in duplicates employing suppressor and thereafter, detecting chloride ions using a conductivity detector. A graph is plotted with amount of chloride (in µg) on X-axis and peak area (AUC) on the Y-axis. From the plots, it was found that chloride standards ranging from 1-8 µg was linear with a regression (R2) = 0.99 with % RSD within 0.4-1.6 % with a back calculated recovery amount ranging from 98-101% for the standard points with the working standard used. (Figure 2, Table 5). The typical chromatographic profile of phosphate will have one major peak at RT ~ 2.3 minutes.
Chloride Standard column load (µg) Average Area (µS*min) Back calculated Amount from graph % Recovery
1 11.07 1.0 101
2 23.97 2.0 98
3 37.40 3.0 98
4 52.00 4.0 101
5 66.19 5.0 101
6 79.95 6.0 101
7 92.71 7.0 100
8 104.41 7.9 99
Table 5
Next, analysis of chloride in test protein sample was performed. For this, sample was diluted 4 to 10 fold in diluent containing 16 mM Histidine in water. 1 mL of diluted protein sample was then loaded onto 30 kDa cut off centrifugal filter and spun at 2900 g for 30-40 minutes. The retentate (containing protein) was discarded and the permeate (containing analyte, which is phosphate in present case) was collected for subsequent analysis. 10-50 µL was injected into and run through a suppressed ion chromatography column at a flow rate of 0.8 – 1.5 mL/min. Mobile phase consisted of 4.5mM Sodium carbonate and 1.4mM Sodium bicarbonate. The sample was eluted from the column under isocratic conditions for a maximum run time of about 8 minutes and detected using a conductivity detector. Based on the chromatographic profile of the multiple test sample runs and comparing the same with the standard curve, average concentration of chloride ions was calculated.
Concentration of chloride (mM) in test sample was calculated using the equation:

A representative data for result of observed chloride ion concentration from 6 different runs of in-house tocilizumab (intravenous; 20 mg/mL) samples (10 fold diluted in 16 mM histidine) is shown in Table 6.

Samples Observed Conc. (mM) in different injections Avg conc. (mM) SD
1 2 3 4 5 6
Tocilizumab (intravenous) 8.7 8.5 8.5 8.5 8.5 8.7 8.5 0.15
Table 6
In case of validation studies, % recovery of anions was calculated using the equation:

Representative data for in-house abatacept samples (5 or 10 fold diluted) and in-house vedolizumab (60 mg/mL) samples indicating % recovery calculated as per above equation are shown in Table 7 & 8 respectively.
Sample Details Centrifugation time (min) Observed Conc. (mM) Expected Conc.
(mM) % Recovery
Abatacept 30 23.17 25 93
40 22.56 92
Table 7
Samples Centrifugation time (min) Area (µS*min) Observed Chloride Conc. (mM) Expected Conc (mM) % Recovery
Vedolizumab_10X dilution_Run1
30 48.074 108.50 117.6 92.3
Vedolizumab_10X dilution_Run2
40 48.413 109.23 117.6 92.9
Table 8

Example 3: Effect of presence/absence of histidine on percentage recovery of analyte
Several attempts to mitigate Donnan effect in the measurement of phosphate and chloride ions were made. For example, three different diluents were used for assessing % recovery: water for injection (WFI), 1 X formulation buffer (FB) without phosphate, 16mM histidine (in WFI). For example, in case of Tocilizumab (20 mg/mL), constitution of 1XFB without phosphate is: 7.43 mM L-Histidine, 8.4 mM L-Histidine Hydrochloride monohydrate, 30mg/mL Sorbitol, 0.5mg/mL (w/v) Polysorbate 80 at pH 6.0. Sample was diluted four folds using different diluents and then processed through centrifugal filters at 2900 x g for 40 min. Representative data may be found in Table 9. Sample diluted with 16mM Histidine could return 4% and 13% higher % recovery when compared to use of 1X FB without phosphate and WFI respectively.
Sample I.D. Diluent Day 1 Day 2 Day 3 Average Expected conc. (mM) % Recovery
Tocilizumab WFI 2.7 2.8 2.9 2.8 3.8
74
1X FB without Phosphate 3.1 3.1 3.3 3.1 82
16mM Histidine 3.3 3.4 3.4 3.3 87
Table 9
Example 4: Effect of dilution fold on percentage recovery of analyte
To find the optimum sample dilution fold, three different sample dilutions were tested. Representative results indicating effect of fold dilution on % recovery of chloride ions in in-house abatacept (25 mg/mL) samples is tabulated in Table 10. All 3 dilution folds gave acceptable % recovery for expected 40mM and 25 mM concentrations.
Sample ID Dilution fold Area µS*min Observed Conc. (mM) Expected Conc. (mM) % Recovery
Abatacept 5 106.73 39.05 40 98
10 89.74 39.55 99
20 43.05 38.95 97
Abatacept 5 61.14 22.7 25 91
10 52.96 23.7 95
20 25.83 24.1 96
Table 10
Example 4: Effect of centrifugation time on percentage recovery of analyte
To assess the effect of centrifugation time on better recovery of analyte, several experiments were conducted. For example, 10 fold diluted test sample (abatacept) was added to the centrifugal filter and spun at 2900 x g for different time point’s viz. 30 minutes, 40 minutes and 50 minutes. Following this, percentage recovery of chloride was calculated. The representative data is given in Table 11. It was consistently observed that centrifugation time of 30 and 40 minutes gave maximum and acceptable ranges of % recovery, while, the same at 50 mins was below acceptance criteria. Hence, 30 mins or 40 mins of centrifugation time was selected for further studies.
Sample Details Centrifugation time (min) Observed Conc. (mM) Expected Conc. % Recovery
(mM)
Abatacept (IV) 30 23.17 25 93
40 22.56 92
50 21.96 88
Table 11

,CLAIMS:CLAIMS

We claim:
1. A method for measuring anion species present in a protein sample, wherein the method comprises:
a) diluting the sample in a solution containing histidine;
b) centrifuging the sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) subjecting the permeate of step b) to suppressed ion chromatography column with a mobile phase comprising sodium carbonate and sodium bicarbonate;
d) collecting the eluate containing the anion species under isocratic conditions;
e) detecting anion species in the eluate using an electrical conductivity detector; and
f) comparing the chromatographic profile of the eluate with that of a standard curve, thereby measuring anion species in the protein sample;
wherein the method mitigates Donnan effect substantially.
2. The method as claimed in claim 1 wherein, the concentration of histidine is 10 mM to 20 mM, preferably 16 mM.
3. The method as claimed in claim 1 wherein, the sample is diluted at least about 5 fold, at least 6 about fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least 15 fold or at least 20 fold; preferably 5 fold or preferably 10 fold, or preferably 20 fold.
4. The method as claimed in claim 1 wherein, the sample is centrifuged using a centrifugal filter at 2900 g for preferably 30 minutes of preferably 40 minutes.
5. The method as claimed in claim 1 wherein, the protein is a monoclonal antibody or a fusion protein.
6. The method as claimed in claim 1 wherein, the anion species are one or more of phosphate or chloride ions.
7. The method as claimed in claim 1 wherein, the mobile phase contains 4.5mM Sodium carbonate and 1.4mM Sodium bicarbonate.
8. The method as claimed in claim 1 wherein, the method mitigates Donnan effect by increasing recovery of anion species by up to at least 13% when compared to using a diluent devoid of histidine.
9. The method as claimed in claim 1 wherein, the protein is vedolizumab or tocilizumab or adalimumab or abatacept.