Description:FIELD OF THE INVENTION
The present invention relates to an analytical method for measuring aminopolycarboxylic acid present in a protein sample by chromatography. More specifically, the invention relates to measuring diethyltriamine tetracetic acid, a chelating agent, in a protein sample by ion-pairing chromatography.
BACKGROUND OF THE INVENTION
Trace heavy metal ions, if present as contaminants in pharmaceutical formulations, can be a cause of serious concern. This is because, they catalyze degradation reactions of the components present, thereby impacting the quality and stability of the drug product. To address this problem, metal ‘chelating agents’ or ‘chelators’ are included in formulations. Chelators sequester metal ions by forming stable complexes with the latter, thereby improving stability and shelf life of the drug product. Commonly used chelators are aminopolycarboxylic acids (APCAs), the most widely used among which being ethylenediamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA or pentetic acid).
During the drug development process, it is important to develop methods that can measure levels of APCAs in the drug formulation, as these methods ensure the stability and quality of the drug product. Direct UV-based detection is challenging as APCAs lack a significant chromophore in their structure. Spectrophotometric detection can be a challenge for protein-based formulations because of interference due to excipients. Considering these, chromatography is a suitable methodology of choice. The state of the art describes gas chromatography (GC), high performance liquid chromatography (HPLC) and ion chromatography (IC), among others, for measuring chelating agents in environmental samples, cosmetics, food products and pharmaceutical formulations. However, existing methods are either time-consuming, or complex in terms of sample processing and methodology or do not have a favorable limit of detection (LOD) for a given type of sample. For example, in the case of GC, so as to make the sample volatile, about a day of sample pre-treatment needs to be done by derivatization of side groups of APCA to form methyl/ethyl/propyl/butyl esters. Also, sample matrix has a huge impact on the accuracy of results during GC. Therefore, despite an LOD of 1µg/mL (for EDTA), GC method is less preferred. Due to lack of chromophore in APCA structure, certain methods require addition of transition metal ions to the sample, leading to formation of a complex between APCA and the metal, thereby facilitating UV detection of the metallocomplexes thus formed. In case of methods employing, reverse phase (RP) HPLC, ion-pair reagents that bind with the analyte are added to the mobile phase, whereby which, the retention of analyte onto the column is enhanced.
Despite existing knowledge, developing a suitable method for a given product involves several critical variables which need to be carefully arrived at for optimal analyte detection. The type and quantity of chelating agent and metal ion used, concentration of counter-ions, pH of the solution, column temperature and presence of other competing matrix components etc. can affect the strength of the APCA-metal ion-counterion complex and the chromatographic run. In the case of protein-based drugs, existing methods teach an additional sample pre-treatment step for precipitating the protein out before APCA detection. Also, as presence of other matrix components such as the protein molecule itself can interfere with UV detection, the method must be customized to temporally separate elution time points of analyte from that of the other components. Also, none of the existing methods teach detection of APCA if present in a protein-sample at very low concentrations such as below about 10 ppm. Therefore, there is a need to develop a simple time-saving method to accurately measure APCA in a protein-based sample with an acceptable lower limit of detection, specifically in the context of therapeutic samples.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a simple, time-saving method for measuring APCAs in a protein sample. More particularly, the method discloses measurement of DTPA in a monoclonal antibody formulation wherein, the method can measure as low as 1.25 ppm DTPA in the sample. The method employs ion-pairing chromatographic separation on a column composed of hybrid material that enhances the peak quality and robustness of the method. The method is not time-intensive, such as requiring APCA derivatization or additional protein removal steps.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Impact of column temperature on measurement of DTPA
Figure 2: Impact of chromatographic column chemistry on peak symmetry
Figure 3: Standard curve generated (AUC vs. Conc. of DTPA in ppm) upon chromatographic separation of working standard solutions as described in Example 4
Figure 4: Peak profile of DTPA in a sample detected at 8.810 minutes upon chromatographic separation as described in Example 4
Figure 5: Specificity of method in the presence of excipients in the sample other than analyte
Figure 6: Linearity of method
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a simple method to measure aminopolycarboxylic acids (“APCA”s) in a protein sample by chromatography.
In an embodiment, the invention discloses a method to measure diethyltriamine pentaacetic acid (“DTPA”) in a protein sample by ion-pairing chromatography, wherein the method can measure as low as 1.25 ppm DTPA in the sample.
In an embodiment, the invention discloses a method for measuring diethyltriamine pentaacetic acid (“DTPA”) in a protein sample, the method comprising:
a) incubating the sample with ferric chloride solution for at least 15 minutes;
b) pre-equilibrating a chromatographic column by passing 80% tetrabutyl ammonium phosphate buffer as mobile phase A and 20% acetonitrile as mobile phase B;
c) injecting the incubate of step a) into the chromatographic column of step b) under an isocratic mobile phase run with 80% tetrabutyl ammonium phosphate buffer as mobile phase A and 20% acetonitrile as mobile phase B;
d) allowing chromatographic separation of DTPA in the column; and
e) measuring DTPA in the flow-through for at least 20 minutes from the time of the injection using a UV detector at 260 nm;
wherein the chromatography column is composed of hybrid monolith material composed of bridged polyethoxysilane;
wherein the chromatographic separation is performed at at least 500C; and
wherein the method can measure as low as 1.25 ppm DTPA in the sample.
In an embodiment, the sample is incubated with ferric chloride solution for 15 minutes, or preferably 30 minutes, or preferably 60 minutes.
In an embodiment, the chromatographic column employs a C18 chemistry.
In an embodiment, concentration of the ferric chloride solution is at least 30 mM.
In an embodiment, concentration of tetrabutyl ammonium phosphate buffer is at least 20mM.
In another embodiment, the chromatographic separation is performed preferably at 650C, or more preferably at 600C.
In an embodiment, DTPA in the flow-through is measured for 20 minutes from the time of the injection using a UV detector at 260 nm.
In another embodiment, DTPA in the flow-through is measured for 30 minutes from the time of the injection using a UV detector at 260 nm.
In an embodiment, the protein is a monoclonal antibody.
In yet another embodiment, the protein is nivolumab.
In yet another embodiment, the protein is ipilimumab.
Definitions:
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.
The term “C18” as used herein refers to octyldecylsilane referring to the number of carbon atoms bound to the stationary phase of the chromatographic column.
The term “hybrid” as used herein includes inorganic-based chromatographic material wherein an organic functionality is integral to the internal and surface aspects of the structure. For example, in a case where the inorganic component is silica, ‘hybrid silica’ refers to chemically modified one or more silica molecules bearing aliphatic and/or aromatic moieties, which may additionally be substituted with desired functional groups. The said desired functional groups may include, but not limited to, alkyl, aryl, cyano, amino, hydroxyl, diol, nitro, ester, ion exchange moieties of polar functional groups.
The term “monolith” as used herein refers to materials that are characterized by a continuous interconnected phase of porous material. The pores, referred to as macropores allow liquid flow with minimal resistance, low back pressures, resulting in highly efficient chromatographic separation.
The term “protein sample” refers to a preparation, such as serum, vaccine, blood and blood components, or recombinant therapeutic proteins derived from animal products or another biological source.
Abbreviations:
AUC - Area under curve
Conc. - Concentration
DTPA - diethyltriamine pentaacetic acid
ppm - parts per million
% RSD - % Relative standard deviation
RT - Retention time
Vol. - Volume
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.
The protein sample used in examples described herein is Nivolumab formulation comprising succinate buffer, sodium chloride, trehalose, methionine, DTPA and polysorbate as excipients. However, it will be obvious to a person skilled in the art that the methodology disclosed herein can be extended to any pharmaceutical drug formulation that comprises DTPA as an excipient.
Example 1: Preparation of standards and samples for run
Working standard solutions
For sample analysis, a standard curve was first plotted using DTPA standard solutions at working concentration ranges of 1.25 to 40 ppm. For this, a stock solution of 800 ppm DTPA was first prepared, from which, an 80 ppm working solution was then made. Following this, working concentrations as in Table 1 and Table 2 were prepared as per the scheme shown:
Sl. No. Working conc. of DTPA solution Vol. of DTPA solution added (µL) Vol. of mobile phase A added (µL)
1 40 ppm 500 µL of 80 ppm 500 µL
2 20 ppm 500 µL of 40 ppm 500 µL
3 10 ppm 500 µL of 20 ppm 500 µL
4 5 ppm 500 µL of 10 ppm 500 µL
5 2.5 ppm 500 µL of 5 ppm 500 µL
6 1.25 ppm 500 µL of 2.5 ppm 500 µL
Table 1
Sl. No. Working conc. of DTPA solution Vol. of DTPA solution added (µL) Vol. of DTPA solution added (µL)
1 15 ppm 200 µL of 20 ppm 200 µL of 10 ppm
2 7.5 ppm 200 µL of 10 ppm 200 µL of 5 ppm
Table 2
Pre-preparation
Pre-preparation refers to processing of sample or standard solutions to allow metallocomplex formation of DTPA with Fe3+ ions. For this, sample or standard (300 µL) was incubated with 10 µL 30 mM FeCl3 solution for at least 15 minutes.
Example 2: Impact of column temperature on measurement of DTPA
In an attempt to select a suitable column temperature that would enable analysis of DTPA at very low concentrations, 1.25 ppm DTPA samples incubated with 30 mM FeCl3 solution as described in Example 1 was run on Symmetry300 C18 column at 300C and 600C. Symmetry300 column is a standard reverse phase HPLC column composed of silica. An isocratic run of mobile phase consisting of 80% (v/v) of 20mM Tetra butyl ammonium (TBA) phosphate buffer as mobile phase A and 20% (v/v) of acetonitrile as mobile phase B was employed. Flow-through was analysed using a photo-diode array detector for measurement of DTPA at 260 nm. Result is depicted in Figure 1.
It was found that the chromatographic run at 600C gave a better tailing factor (T) of 1.98 compared to a the run at 300C (tailing factor - 4.19). Therefore, 600C was selected as a suitable temperature for the chromatographic run.
Example 3: Impact of chromatographic column chemistry on peak symmetry
During chromatographic separation, the ideal peak in the chromatogram, corresponding to that of the analyte should be symmetric on both sides, indicating precision of the method. Tailing factor (T) is a measure of peak symmetry, which should be unity (1) for perfectly symmetrical peaks and increases as the less desirable ‘tailing’ becomes more pronounced. Tailing factor is calculated as:
T = (a+b)/2a
where, a and b are the peak half-widths at 5% of the peak height.
In an attempt to select a suitable column that would give a desirable peak symmetry (i.e, closer to unity), two different C18 columns were compared: Symmetry300 C18 column and Xbridge peptide BEH C18 column (WatersTM). Symmetry300 column is a standard reverse phase HPLC column composed of silica, while XbridgeTM BEH C18 is a reverse phase HPLC column composed of hybrid monolith material composed of bridged polyethoxysilane. Sample preparation was performed as described in Example 1. Samples were run on the two different columns under the conditions described in Example 4.
Table 3 shows tailing factor after the chromatographic runs using protein sample containing 1.25 ppm DTPA. Figure 2 gives a qualitative representation of the same. It was found that separation using XbridgeTM BEH C18 column gave an acceptable tailing factor of 0.99, closer to the acceptance criteria compared to Symmetry300 column even at a very low concentration of DTPA (1.25 ppm).
Column details Tailing factor (T)
1 Symmetry300 C18 5µm column 1.98
2 XbridgeTM BEH C18 column 0.99
Table 3
Example 4: Analysis of DTPA in sample
Analysis of DTPA in sample was performed by ion-pairing chromatography. Mobile phase A and mobile phase B used for chromatographic separation were 20mM Tetra butyl ammonium phosphate buffer and acetonitrile respectively in an isocratic run at a ratio of 80:20 (v/v).
Following pre-preparation, sample(s) and standards were loaded onto XBridge™ BEH300 C18 ((5 µm, 4.6 mm * 250 mm; WatersTM) for chromatographic separation. Column temperature was maintained at 600C. An isocratic run of mobile phase consisted of 80% (v/v) of 20mM Tetra butyl ammonium (TBA) phosphate buffer as mobile phase A and 20% (v/v) of acetonitrile as mobile phase B. Flow-through was analysed using a photo-diode array detector for measurement of DTPA at 260 nm. Results are depicted in Table 4 and Figures 3 to 4.
A DTPA percentage recovery of 93-105% was obtained using methodology described herein. The DTPA standard of 1.25 ppm gave a recovery of 105% which is the limit of detection of this method.
DTPA Standard Conc. (ppm) AUC Back calculated conc. (ppm) % Recovery
1.25 37658 1.31 105
2.5 72435 2.31 93
5 166991 5.05 101
10 338340 10.01 100
15 519700 15.26 102
20 676517 19.80 99
Table 4
Example 5: Specificity of method
Specificity is the ability to assess unequivocally the analyte in the presence of components which may expected to be present in the sample. As the multiple excipients present in the drug formulation can interfere with the analyte signal, it is critical to analyze specificity of the method. For evaluating specificity of disclosed method and thereby to assess ability of disclosed method to mitigate interference due to signal from excipients, all the excipients were analyzed by methodology described in Example 4.
Results are depicted in Figure 5. It was found that signal from excipients was well separated out compared to signal due to DTPA on the chromatogram. Hence, it was inferred that there was no matrix interference, indicating that the method is specific to DTPA.
Example 6: Linearity of method
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.
For assessing linearity of method, working standards at varying concentrations were prepared. 300uL of working standards were incubated with 10uL of 30 mM FeCl3 solution and run on chromatographic method as described in Example 1. Results are depicted in Table 4 and Figure 6.
Standard Conc. (ppm) AUC Back calculated conc. (ppm) % Recovery RT (min)
2.5 84716 2.52 101 8.948
5 171225 4.93 99 8.956
7.5 264885 7.54 101 8.993
10 356091 10.08 101 8.983
12.5 439214 12.39 99 8.972
15 534205 15.04 100 8.974
Table 5
Example 7: Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
To assess robustness of the method, varying sample volumes (200 µL – 400 µL) were incubated with 10 µL 30 mM FeCl3 solution. Results are depicted in Table 6. It was found that the method gave robust and reliable results for sample volumes in the aforementioned ranges.

Sample Theoretical Conc. (ppm) AUC Back calculated conc. (ppm) % RSD % Recovery Retention time (min)
200ul sample +10ul FeCl3 8 345998 9.80 2.3 122 9.046
300ul sample +10ul FeCl3 8 360112 10.19 127 9.056
400ul sample +10ul FeCl3 8 361103 10.22 128 9.138
Table 6
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments and examples are therefore to be considered in all respects illustrative rather than limiting the invention described herein.
, Claims:CLAIMS
We claim:
1. A method for measuring diethyltriamine pentaacetic acid (“DTPA”) in a protein sample, the method comprising:
a) incubating the sample with ferric chloride solution for at least 15 minutes;
b) pre-equilibrating a chromatographic column by passing 80% tetrabutyl ammonium phosphate buffer as mobile phase A and 20% acetonitrile as mobile phase B;
c) injecting the incubate of step a) into the chromatographic column of step b) under an isocratic mobile phase run with 80% tetrabutyl ammonium phosphate buffer as mobile phase A and 20% acetonitrile as mobile phase B;
d) allowing chromatographic separation of DTPA in the column; and
e) measuring DTPA in the flow-through for at least 20 minutes from the time of the injection using a UV detector at 260 nm;
wherein the chromatography column is composed of hybrid monolith material composed of bridged polyethoxysilane;
wherein the chromatographic separation is performed at at least 500C; and
wherein the method can measure as low as 1.25 ppm DTPA in the sample.
2. A method as claimed in claim 1 wherein, the sample is incubated for preferably15 minutes, or preferably 30 minutes, or preferably 60 minutes.
3. A method as claimed in claim 1 wherein, the column employs a C18 chemistry.
4. A method as claimed in claim 1 wherein, concentration of the ferric chloride solution is at least 30 mM.
5. A method as claimed in claim 1 wherein, concentration of the tetrabutyl ammonium phosphate buffer is at least 20mM
6. A method as claimed in claim 1 wherein, the chromatographic separation is performed preferably at 650C, or more preferably at 600C.
7. A method as claimed in claim 1 wherein, the DTPA in the flow-through is measured for 20 minutes from the time of the injection using a UV detector at 260 nm.
8. A method as claimed in claim 1 wherein, the DTPA in the flow-through is measured for 30 minutes from the time of the injection using a UV detector at 260 nm.
9. A method as claimed in claim 1 wherein, the protein is a monoclonal antibody.
10. A method as claimed in claim 9 wherein, the monoclonal antibody is nivolumab, pembrolizumab or ipilimumab.