DESC:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL / COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION: “Method for detection and quantitation of non-ionic surfactant in a therapeutic preparation”
2. APPLICANT(S)

(a) NAME: Dr. Reddy’s Laboratories Limited
(b) NATIONALITY: Indian
(c) ADDRESS: 8-2-337, Road No. 3, Banjara Hills,
Hyderabad, Telangana,
India-500 034
3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL SPECIFICATION
The following specification describes the invention. COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it is to be performed.
4. DESCRIPTION (Description shall start from next page)

Description starts from next page.

5. CLAIMS
Claims are enclosed in a separate page

6. DATE AND SIGNATURE
Given at the end of last page of specification
7. ABSTRACT OF THE INVENTION
Given at a separate page.

FIELD OF INVENTION
The present invention relates to the field of analytical testing of therapeutic preparations. Particularly, the invention describes a method for quantitation of non-ionic surfactants in biotherapeutic preparations with reduced interference from protein molecules.
BACKGROUND
Surfactants or ‘surface active agents’ lower the interfacial tension between a solution and a different phase (e.g. between two liquids, a gas and a liquid, or between a liquid and a solid). Non-ionic surfactants consist of a hydrophilic head group and a hydrophobic tail, are without any charge and are relatively non-toxic. Examples of non-ionic surfactants, are Polysorbate 20, Polysorbate 80, Triton X-100 and Poloxamer-188.
Polysorbate is a hydrophilic non-ionic surfactant formulated by the reaction of sorbitan fatty acid ester (a non-ionic surfactant) with ethylene oxide. Polysorbate 80 (PS80) and polysorbate 20 (PS20) are also known as Tween® 80 and Tween® 20 respectively. The fatty acid hydrocarbon chain and the polyoxyethylene sorbitan group provide respectively hydrophobic and hydrophilic characters to the polysorbate molecule. Amongst other non-ionic surfactants, PS80 is one of the most commonly used non-ionic surfactants in formulations of biotherapeutics.
Polysorbate 80 is an excipient used in biotherapeutic preparations to prevent surface adsorption and stabilize proteins against aggregation induced by stresses such as agitation and shear in aqueous formulations of biotherapeutic preparations. PS80 is used as an excipient in pharmaceutical compositions because of its effectiveness at low concentrations, relative low toxicity, and ability to not only inhibit protein surface adsorption and aggregation under various processing conditions but also acts as a stabilizer against protein aggregation.
Triton X-100 is another non-ionic surfactant which has a lipophilic or hydrophobic aromatic hydrocarbon group and a hydrophilic polyethylene oxide chain. It is used in pharmaceuticals to inactivate some lipid enveloped viruses like HBV. Poloxamer, also a non-ionic surfactant, is additionally used in cell culturing as it provides cushioning effects to cells, thereby contributing to lesser shearful stress.
In case of complex biotherapeutic products, quality of the product is critical for protein stability and efficacy. For quality control purposes, it is mandatory to determine the concentration of non-ionic surfactants in the final drug substance (DS) and drug product (DP). This is because non-ionic surfactants contain residual amounts of peroxides which can induce oxidation in the therapeutic protein. Oxidation leads to degradation of protein that can affect drug potency over time and may reduce shelf life. For protein pharmaceuticals, oxidation is one of the major chemical degradation pathways.
In order to analyse non-ionic surfactants, a highly sensitive method is desired. Various methods are mentioned in literature that can be used for quantitation of non-ionic surfactants, specifically polysorbate. These methods include fluorescence polarization (FP) assay, RP-HPLC-UV analysis of lauric/oleic acid, Capillary Electrophoresis (CE) and RP-HPLC-CAD. Some of these methods are deficient in terms of sensitivity, accuracy, specificity, and throughput.
Further, shelf life of any therapeutic protein sample is closely related to the thermal stability of a substance which is added as an excipient, which includes the surfactant. A formulation is comparatively more thermally stable, if the desired properties are unchanged over a larger range of temperature that the formulation may be practically exposed to. Without thermal stability studies, the temperature at which the substance starts to decompose or react cannot be determined. It is desirable that an analytical method displays consistent performance in assessing thermal stability of a solution over a larger range of temperatures.
Present invention describes a sensitive, accurate and reproducible method to quantitate and detect non-ionic surfactant present in biotherapeutic preparation with a lower limit of quantitation as low as 0.1µ. The disclosed method facilitates rapid quantitation and accurate detection of non-ionic surfactants present in protein sample with reduced interference from protein molecules. The invention also works for evaluation of thermal stability study of non-ionic surfactant.
SUMMARY OF THE INVENTION
Accordingly, the present invention discloses an analytical method for quantitation, analysis and detection of a non-ionic surfactant in a protein-based biotherapeutic preparation. The method employs mixed mode anion exchange HPLC column to quantitate non-ionic surfactant and uses aerosol detector for detection. Aerosol detectors convert chemicals in the sample into aerosol particles which are then detected by an electrometer. Aerosol detectors are used in conjunction with HPLC and ultra-high performance liquid chromatography (UHPLC). Both CAD and ELSD exhibit non-linear responses for most sample types, however over small ranges (e.g. 1 - 100 ng), CAD response is reasonably linear for non-volatile analytes with detection limit 1 - 3ng. The charged aerosol detectors are used for the analysis of therapeutic proteins due to the flexibility and performance of CAD.
The detection method further employs stream valve application which avoids detector fouling. The method described in present invention enables analysis in a single experiment without the need for sample processing and dilution, whereby which, the method is time saving. By using the method disclosed herein, it is possible to perform thermal stability studies of non-ionic surfactants stored in different temperature conditions with superior quality results. Using this method, it has been demonstrated that PS80 (a non-ionic surfactant) levels as low as 0.1 µg in drug preparations can be detected and quantified. Thus, the method is also suitable for determining the quality of non-ionic surfactants, be it in the original stock or dispensed stock solution, or degradation due to time or storage conditions.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Standard curve of Polysorbate 80 standards for linearity
Figure 2. Overlaid peak profiles of DRL mab with PS 80, DRL mab without PS 80, PS 80 standard and MilliQ®
Figure 3. Overlay chromatogram of PS80 with and without stream valve application
Figure 4. Overlay of polysorbate 80 peak profile for 18 days sample stored at 37°C, 2 – 8°C and freshly prepared sample for comparison
Figure 5. Overlaid peak profiles of Triton X-100 standard, sample with Triton X-100 and sample without Triton X-100
Figure 6. Overlaid peak profiles of Poloxamer 188 standard, sample with Poloxamer 188 and sample without Poloxamer 188
DETAILED DESCRIPTION
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. Any methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present invention.
Various embodiments of the disclosed invention discloses a method for quantitation of a non-ionic surfactant present in a solution.
In an embodiment the claimed invention describes an analytical method of detecting and quantitating a non-ionic surfactant in a solution comprising:
a. loading the solution onto a mixed mode anion exchange chromatography column;
b. eluting the non-ionic surfactant using mobile phase A and mobile phase B, wherein, mobile phase A comprises an acid with water for injection (WFI) and mobile phase B comprises an acid with an organic solvent; and
c. detecting and quantifying the non-ionic surfactants using an aerosol detector;
wherein the method can be performed for stability studies of sample stored at a wider range of temperatures; and
wherein the method optionally uses stream valve application in integration with the aerosol detector.
In an embodiment, the solution is a biotherapeutic preparation or a stock solution of non-ionic surfactant.
In another embodiment the analysis of non-ionic surfactant is carried out using mixed mode anion exchange chromatography in HPLC system.
In yet another embodiment the mixed mode anion exchange chromatography uses two mobile phases: mobile phase A and mobile phase B, wherein mobile phase A comprises formic acid in water and mobile phase B comprises formic acid in acetonitrile.
In yet another embodiment mobile phase A comprises 0.2 - 2% formic acid in water for injection (WFI).
In yet another embodiment mobile phase B comprises 0.2 - 2% formic acid in organic solvent wherein the organic solvent is acetonitrile.
In yet another embodiment the column temperature is 30°C and column flow rate is 1mL/min.
In yet another embodiment the aerosol detector used for detection is selected from Charged Aerosol Detector (CAD) and evaporative light scattering detectors (ELSD).
In yet another embodiment the non-ionic surfactant is Polysorbate 20, Polysorbate 80, Triton X-100 or Poloxamer 188.
In yet another embodiment concentration of non-ionic surfactant in the therapeutic preparation is in range of 0.07% to 1 % w/v.
In one embodiment the samples used for analyzing thermal stability are stored at temperature conditions ranging from about 2 to about 37°C.
In yet another embodiment the non-ionic surfactant is detected and quantified at a time period of up to about 40 days.
Definitions
The term “non-ionic surfactant” as used herein this invention refers to a surface active agent consisting of a hydrophilic head group and a hydrophobic tail, carry no charge and are relatively non-toxic. Examples of non-ionic surfactants are Polysorbate 20, Polysorbate 80, Triton X-100 and Poloxamer.
The term “analytical method” herein this invention refers to a chromatographic method i.e. mixed mode anion exchange chromatography to analyze and quantitate the non-ionic surfactant and biotherapeutic preparation.
The term “solution” herein refers to a biotherapeutic preparation or a stock solution of non-ionic surfactant.
The term "biotherapeutic" (or interchangeably, “therapeutic” ) herein is used in the broadest sense and it covers proteins that are genetically engineered through recombinant DNA technology, which are of therapeutic significance in the treatment of ailments. Biotherapeutics include monoclonal antibodies, fusion proteins, polyclonal antibodies, multispecific antibodies and antibody fragments so long as they exhibit the desired biological activity.
The term “biotherapeutic preparation” herein refers to a composition or formulation of biotherapeutic molecules or fragments thereof that is produced by mammalian cell culture.
The term “mobile phase” used in this invention refers to the liquid or gas that flows through a chromatography system, moving the materials to be separated at different rates over the stationary phase.
The term “gradient” used herein refers to the conditions used for getting separate and clear peak of non-ionic surfactant amongst all content present in the biotherapeutic composition.
The term “stability” used herein refers to ability of a substance to remain unchanged over time under stated or reasonably expected conditions of storage and use.
The term “DRL mab” used herein refers to the test therapeutic monoclonal antibodies or therapeutic protein.
EXAMPLES
Example 1: Preparation of Mobile Phases and Standard
Mobile phase A was prepared by adding 800 mL of water for injection (WFI) in a 1 litre volumetric flask to which 2 ml of Formic acid was added and the solution was made up to 1 litre by addition of water for injection. Mobile phase B was prepared by adding 800 mL Acetonitrile in a 1 litre volumetric flask to which 2 ml of Formic acid was added and the solution was made up to 1 litre by addition of acetonitrile.
Standard stock solutions of 2 mg/mL PS80, PS20, Triton X-100 and Poloxamer 188 were prepared using water for injection and 0.2 mg/mL working standards for each surfactant were prepared using standard stock solutions.
Example 2: Linearity of system
Standard curve was generated by injecting different amount of working PS80 (0.2mg/mL) as listed in Table 1. Based on the better peak profile observed, linearity of the polysorbate 80 working standard from 1 µg column load to 12 µg column load and neat sample was checked. The back calculated recovery values ranged from 96 – 102% and R2 value was 1.00. Figure 1 shows linear standard curve.
Column load (µg) AUC
(pA*sec) Injection volume (µL) Back calculated conc. (µg) % Recovery
1 120177626 5 0.9 95
2 206068497 10 1.9 97
4 396206916 20 4.2 104
6 551904389 30 6.0 100
8 722878083 40 8.0 100
10 900997221 50 10.0 100
12 1065988651 60 12.0 100
Table 1: PS80 content % recovery at different volume and concentration
Example 3: Precision
Repeatability/Intra assay precision: Six injections of neat sample were injected and evaluated for intra-assay precision. 0.1 mg/mL PS80 was prepared and 50 µL injection volume was injected in system such that 5 µg load should enter in column. The % RSD of PS80 quantitated in the test samples between the six injections was = 2% for intra-assay precision.
Sample ID Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Injection 6 Expected
Conc. (mg/mL) Average SD %RSD
DRL_01 0.48 0.47 0.47 0.47 0.47 0.47 0.5 0.47 0.004 0.9
DRL_02 0.37 0.37 0.38 0.36 0.37 0.36 0.5 0.37 0.007 1.8
DRL_03 0.159 0.16 0.156 0.158 0.163 0.158 0.2 0.16 0.003 1.6
DRL_04 0.22 0.23 0.23 0.23 0.23 0.23 0.2 0.23 0.005 2
Table 2: PS80 content % RSD of in test samples for intra assay precision
Example 4: Robustness
a. Column temperature change: Column temperature as per method is 30°C but to assess the robustness of method, column temperature was changed to ±2°C of actual temperature to see the effect of PS 80 in samples. Table 3 shows that change in column temperature does not have any significant effect on area under curve of PS80 samples. The samples showed RSD <3% for the AUC values.
AUC (pA*sec)
Sample ID 28°C 30°C 32°C Average SD % RSD
DRL_02 414971889 429083386 409054631 417703302 10289957 2.5
DRL_03 359321614 372382973 365666558 365790382 6531560 1.8
Table 3: Summary of % RSD for column temperature change
b. Mobile phase stability: Prepared mobile phase was used for 4 consecutive days. The standard AUC was checked on Day 1 and Day 4 for comparison. Table 4 shows that there was less than 5% RSD for column load 4 - 12 µg and 12 -16% RSD for column load 1 - 2 µg.
Average AUC (pA*sec)
Column load (µg) Day 1 Day 4 Average SD %RSD
1 170340061 147098180 105812747.2 16434492 15.5
2 272524702 243021708 171848803.8 20861767 12.1
4 464225959 444743351 302989771.3 13776284 4.5
6 667903874 671072427 446325435.5 2240506 0.5
8 870774691 867024262 579266320.2 2651953 0.5
12 1255695701 1217840935 824512215.8 26767362 3.2
Table 4: Mobile phase stability
Example 5: Specificity
For evaluating the interference of these excipients, formulation buffer without polysorbate 80, water for injection, polysorbate 80 standard and DRL mab formulation buffer was analysed in the same method conditions to check the interference.
Figure 2 shows that the polysorbate 80 standard and DRL mab formulation buffer shows polysorbate 80 peak at retention time 5.06 minutes. The water for injection and DRL mab formulation buffer without polysorbate 80 shows similar non-significant peak at 5 minutes retention time which is due to high sensitivity of detector which proves that DRL mab without polysorbate 80 shows minimal interference which is equal to water for injection. The blank injection has an interfering peak at the same retention time of PS20 and PS80 which is caused by the mobile phase gradient change.
Example 6: System suitability
0.1 mg/mL PS80 was prepared and 50 µL injection volume was injected in system such that 5 µg load should enter in column. The system suitability was carried out by two analyst on two different days. Table 5 shows the working system suitability data, where in the % RSD between duplicate injections is <1%, % recovery is within 93 – 101, USP tailing is 2.1 and plate count is >3000.
Sample ID AUC Average AUC
(pA*sec) % RSD Calculated observed conc. From graph (µg) Injection volume (µL) Observed Conc. (µg/µL) observed Conc. (mg/mL) Expected conc. (mg/mL) % Recovery Asym @ (10)^2 USP Plate Count USP Tailing
S_01 481128055 482918043 0.5 5.1 50 0.1 0.1 0.1 101 8.8 3126.4 2.1
S_02 484708030 8.2 3823.7 2.1
S_03 447075215 444644654 0.8 4.6 50 0.09 0.09 0.1 93 9.1 3171 2.1
S_04 442214093 8.2 3360.3 2.1
Table 5: System suitability
Example 7: Injection and running of samples
Column used for running of the sample was Waters Oasis Max column with dimensions 2.1 X 20 mm and 30 µm particle size which is a reversed phase column and used in strong anion exchange chromatography. For column cleaning and regeneration column inlet was connected to column without connecting column outlet to CAD, and mobile phase B was passed. After column cleaning and regeneration, column was equilibrated by connecting column outlet to CAD.
Blank samples of water for injection were injected before PS80 standard and test sample. Further 0.2 mg/mL polysorbate 80 standard and test sample were injected into the column. Sample analysis was done by injecting 10 µL and 20 µL of test sample in duplicates. After analysis, run column was conditioned at 1.0 mL/min.
Example 8: Stream valve (liquid On/Off) Application
As protein elutes at void volume its high concentration can deteriorate life of detector. Stream valve option passes first few minutes to drain such that the protein fraction enters drain and only the polysorbate fraction enters the detector. Method was enhanced in instrument by adding liquid flow (Off/On) parameter in time table option. Figure 3 shows overlaid chromatogram of PS80 for stream valve application on and off.
Example 9: Thermal stability of polysorbate 80
Above method was checked for its stability indicating parameter, wherein 2 mg/ml polysorbate 80 samples were kept for study at 2-8°C and 37°C for 13 days, 18 days and 40 days, for each time interval the samples were checked along with the freshly prepared 2 mg/mL polysorbate 80. All the samples were diluted by 10 folds with water for injection and 0.2 mg/mL sample was injected to check peak profile. Figure 4 shows overlay of polysorbate 80 peak profile kept for 18 days at 37°C, 2-8°C and freshly prepared sample for comparison. The peak profile in Figure 4 shows that polysorbate 80 sample was stable after 18th day when it was stored at 2-8°C but shows 62% loss of PS 80 content when stored at 37°C.
Freshly prepared PS 80 when compared to PS 80 stored at 2-8°C for 40 days showed minimal loss of 3 percent. Thus, the method is capable to capture the stability parameter of the PS 80 due to temperature excursion.
Example 10: Sample analysis data and peak profiles of Triton X-100
In order to assess the method performance, triton X-100 standards were subjected to run in the sequence and to generate standard curve between concentration (in µg) versus area under curve (in pA*sec). The samples were injected neat into the column. The results are tabulated in Table 6 and showed good regression and recovery for standards ranging from 1 µg - 12 µg. The coefficient of determination i.e. R2 was 1.00 whereas % RSD between duplicate injection < 1.5 and back calculated recovery within 93% - 103%.
Column load of Triton X-100 (µg) AUC (pA*sec) Average AUC (pA*sec) %RSD Injection volume (µL) Back calculated conc. (µg) % Recovery
1 118816964 118390653 0.51 5 0.93 93
117964342
2 179639263 181304570 1.30 10 1.92 96
182969876
4 321258818 317983233 1.46 20 4.06 102
314707648
6 452463499 452008672 0.14 30 6.17 103
451553844
8 570907138 569413734 0.37 40 8.01 100
567920330
10 690949132 696203831 1.07 50 10.01 100
701458529
12 810452639 816626320 1.07 60 11.90 99
822800001
Table 6: Standard curve data for Triton X-100
Sample ID AUC Calculated observed conc. From graph (µg) Injection volume (µL) Observed Conc. (µg/µL) Observed Conc. (mg/mL) Expected conc. (mg/mL) % Recovery
WFI 65956729 0.1 40 0.00 0.003 0.1 NA
AB-BR-091,096 OUTPUT EXP2 345194883 4.5 40 0.11 0.112 0.1 112
AU00917 TFFII LOAD 83089369 0.4 40 0.01 0.009 0.1 NA
AB-BR-107 NTEL 256022921 3.1 40 0.08 0.077 0.1 77
Triton x-100 _0.1mg/ml 346758701 4.5 40 0.11 0.113 0.1 113
ABBR087 DAY13 NTEL 283257612 3.5 40 0.09 0.088 0.1 88
Table 7: Summary of data observed for samples
The experiment performed using parameters as mentioned in previous examples showed good standard curve data and were checked for DRL mab in-process samples and the results show absence of triton X-100 in TFFII load sample while its presence in NTEL samples (table 7). Figure 5 of chromatographic overlay show similar peak profiles for NTEL sample with that of triton X-100 standard and TFFII load sample showed similar peak profile as that of WFI.
Example 11: Sample analysis data and peak profiles of Poloxamer 188
Poloxamer 188 standard of 0.2 mg/mL concentration was injected with different injection volumes to generate standard curve between concentration (in µg) versus area under curve (in pA*sec). The samples were injected neat into the column. The results are tabulated in Table 8 and showed good regression and recovery for standards ranging from 1 µg - 12 µg. The coefficient of determination i.e. R2 was 1.00 and back calculated recovery was within 91% - 102%.
Sample ID Column load (µg) AUC (pA*sec) Injection volume (µL) Back calculated conc. (µg) % Recovery
Standard (0.2 mg/mL) 1 68867795 5 0.9 91
2 107501479 10 2.0 98
4 181921975 20 4.0 100
6 260402663 30 6.1 102
8 334113531 40 8.1 102
10 404488292 50 10.1 101
12 468732385 60 11.8 98
Table 8: Standard curve data for poloxamer 188
Sample ID AUC Calculated observed conc. from graph (µg) Dilution factor Injection volume (µL) Observed Conc. (µg/µL) Observed Conc. (mg/mL) Expected Conc. (mg/mL) % Recovery
AB SC FB_10X 321102828 7.8 10 10 7.79 7.79 8 97
AB SC FB_20X 185101641 4.1 20 10 8.15 8.15 8 102
DAB-PB-006-20 FDS_10X 312662224 7.6 10 10 7.56 7.56 8 94
DAB-PB-006-20 FDS_20X 186899408 4.1 20 10 8.25 8.25 8 103
Table 9: Summary of data observed for samples
The experiment performed using parameters as mentioned in previous examples showed good standard curve data. Also same system parameters were checked for DRL mab samples are tabulated in table 9. Figure 6 shows chromatographic overlay peak profiles for DRL mab sample with WFI and poloxamer 188 standards.

,CLAIMS:CLAIMS
We claim,
1. An analytical method for detection and quantitation of a non-ionic surfactant in a solution comprising:
a. loading the solution onto a mixed mode anion exchange chromatography column;
b. eluting the non-ionic surfactant using mobile phase A and mobile phase B, wherein, mobile phase A comprises an acid with water for injection (WFI) and mobile phase B comprises an acid with an organic solvent; and
c. detecting and quantifying the non-ionic surfactants using an aerosol detector;
wherein the method can be performed for stability studies of sample stored at a wider range of temperatures; and
wherein the method optionally uses stream valve application in integration with the aerosol detector.
2. The method as claimed in claim 1 wherein the acid used in mobile phase A and B is formic acid and organic solvent used in mobile phase B is acetonitrile.
3. The method as claimed in claim 1, wherein the solution is a biotherapeutic preparation or a stock solution of non-ionic surfactant.
4. The method as claimed in claim 1, wherein the column temperature is 30°C and column flow rate is 0.5 - 1mL/min.
5. The method as claimed in claim 1, wherein the mobile phase A and B comprises 0.2 - 2% formic acid.
6. The method as claimed in claim 1, wherein the detector used for detection of non-ionic surfactant is an Aerosol Detector and selected from Charged Aerosol Detector (CAD) and evaporative light scattering detectors (ELSD).
7. The method as claimed in claim 1, wherein the stream valve application preferentially allows the fraction containing the non-ionic surfactant to pass through detector.
8. The method as claimed in claim 1, wherein the non-ionic surfactant is Polysorbate 20, Polysorbate 80, Triton X-100 or Poloxamer 188.
9. The method as claimed in claim 1, wherein concentration of non-ionic surfactant in the therapeutic preparation is in range of 0.07% to 1 % w/v and wherein the non-ionic surfactant is quantified at a time period for up to 40 days from date of preparation of the solution.
10. The method as claimed in claim 1, wherein the temperature conditions used for stability study are about 2 to about 37?.