DESC:FIELD OF THE INVENTION
The present invention relates to a method for determination of excipients in biologic preparations using chromatography.
BACKGROUND OF THE INVENTION
Sugars and sugar alcohols (or, polyols), present as excipients in biopharmaceutical preparations stabilize proteins against unfolding and aggregation, thereby preserving the protein’s native structure. This is achieved by immobilizing the protein in a rigid, amorphous glassy sugar matrix and by doing so, drastically slowing down degradation (vitrification theory). During lyophilization, the hydroxyl groups of sugars form hydrogen bonds between water and protein by which, the protein’s native conformation is maintained (water replacement theory). In solutions, sugars also help in adjusting osmolality of the formulation. Examples of sugars and polyols widely used as excipients in biopharmaceutical preparations are sorbitol, mannitol, glucose, sucrose, trehalose, and maltose.
Sugars can be present as an excipient in the finished drug product (as mentioned above) or can be present as residual impurities in pre-formulation drug samples. Such residual impurity, primarily originating from raw material sources can potentially cause undesired protein glycation (via Maillard reaction) or aggregation, thereby significantly impacting protein stability. For example, incubation of a glycation-sensitive antibody with 5% glucose at 370C for 5 days was found to increase the glycation product to 80% of the total protein. (Miller, A. K., Hambly, D. M., Kerwin, B. A., Treuheit, M. J., & Gadgil, H. S. (2011). Characterization of site-specific Glycation during process development of a human therapeutic monoclonal antibody. Journal of Pharmaceutical Sciences, 100(7), 2543-2550. https://doi.org/10.1002/jps.22504). Thus there is a need to analyzing drug samples for presence of these entities from time to time to ensure quality of the finished drug product.
Several methods are known in the art for determination of sugars and/or sugar alcohols in protein formulations. These include gas chromatography (GC), Liquid chromatography (LC), Gas liquid chromatography (GLC) or high-performance liquid chromatography (HPLC) coupled with detection methods such as amperometry, mass spectrometry (MS), Flame ionization detector (FID), refractive-index detector (RID) or evaporative light scattering detector (ELSD). Among these, HPLC coupled with determination using RID or ELSD are a preferred choice, it being comparatively economical, simple, and fast. HPLC analysis of sugars is usually performed on amino-bonded silica gel columns. However, the efficiency of these columns is known to decline rapidly with each use, possibly due to loss of the bonded amino groups.
Although analytical methods can be customized to detect a given type or subset of sugar(s), rarely does an existing method teach how to simultaneously differentiate a larger range of possible sugars and polyols present in a protein formulation. For example, existing methods of analysis give overlapping peaks for signals corresponding to maltose, trehalose and sucrose thus, not efficiently differentiating these sugars by a single method. The reasons for this inefficient differentiation can be multifarious including binding chemistry of the analyte entity with the chromatography column and solubility of the entities, among others. Besides, there exists scanty literature on simultaneous analysis of sugars and sugar alcohols in protein-based biopharmaceutical preparations. This can be particularly challenging when a given preparation contains multiple sugars at the same time. In addition, it is beneficial from a logistics angle to have a platform method to analyze several preparations of the same or of different protein molecule(s) without having to make major changes in the method parameters.
It should be noted that although simultaneous analysis may have been reported elsewhere, so far there exists no report of an HPLC-RID based method that can simultaneously analyze glucose, sucrose, trehalose, maltose, sorbitol, and mannitol - the sugars and polyols most often found in protein-based drug preparations. Therefore, there is a need to develop a method for simultaneous analysis of these entities in the said preparation.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a fast, accurate, simple, and reproducible, method based on chromatography for simultaneous analysis of sugars and sugar alcohols, particularly, glucose, sucrose, trehalose, maltose, sorbitol, and mannitol present in biopharmaceutical preparations. The method employs chromatographic separation using column packed with polyamine bonded polymer gel followed by RID-based detection. The said gel is chemically stable, and effectively eliminates issues relating to declining elution times and short service life associated with commonly used cyano- or amino-bonded silica-based columns. The gel is stable over a wide range of pH and is also free from problems of gel shrinkage and swelling associated with conventional polymeric gels. Saccharide analysis can be performed with the disclosed method in a single run with minimal sample preparation and with acceptable precision and sensitivity. Disclosed method will serve as a useful tool for routine analysis of monosaccharides, disaccharides, and sugar alcohols for monitoring profile of analytes and for impurity determination in biopharmaceutical preparations.
BRIEF DESCRIPTION OF THE DRAWINGS

Figure1: Representative chromatograms showing retention time (RT) vs. millivolts (mV) plot of the standard sugar mixture sample with Shodex® EP SC1011-7F column
Figure 2: Representative chromatograms showing retention time (RT) vs. millivolts (mV) plot of the standard sugar mixture sample with Shodex®NH2P series,VG-50 column
Figure 3: Representative chromatograms (retention time (RT) vs. millivolts (mV)) obtained for sample run at three different column temperatures, viz., 300C, 400C and 500C.
Figure 4: Representative overlay of chromatogram for sample run at two different ratios of acetonitrile: water (75:25 and 70:30)
Figure 5: Overlay of the chromatograms obtained after run with test samples (DRL_A#1, DRL_A#2) and with that of other components as shown.
DETAILED DESCRIPTION OF THE INVENTION
Sugars and sugar alcohols (or, polyols) are present as excipients in biopharmaceutical preparations and can also be detected as residual impurities in pre-formulation drug samples. Analyzing the samples for presence of these entities from time to time is necessary for ensuring the quality of the finished drug product. Existing methods are inefficient in simultaneously differentiating sugars and sugar alcohols that are mostly used as excipients in biopharmaceutical preparations, particularly, glucose, sucrose, trehalose, maltose, sorbitol, and mannitol.
The present invention relates to a method for determination of sugars and sugar alcohols in a protein sample using chromatography. More particularly, present method discloses a method for simultaneously determining glucose, sucrose, trehalose, maltose, sorbitol, and mannitol present in the protein sample
In an embodiment, the invention discloses a method a method for determination of one or more of sugars or sugar alcohols present in a protein sample, wherein the method comprises:
a) diluting the protein sample in water;
b) centrifuging the diluted sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) loading the permeate onto a chromatographic column packed with polyamine bonded polymer gel at a defined column temperature;
d) eluting the sugars and sugar alcohols bound to the column using an eluent comprising acetonitrile by isocratic elution at a defined flow rate and the defined column temperature;
e) detecting one or more of the sugars or sugar alcohols using a refractive index detector wherein temperature of the detector is preferably at 400C;
f) determining one or more of the sugars or sugar alcohols present in the protein sample by comparing output with reference to that of a standard mixture.
In an embodiment, the sample temperature is 50C, 60C, 70C, 80C, or more preferably 50C.
In an embodiment, the sample is diluted 2 fold, 3 fold, 4 fold, 5 fold, or more preferably 5 fold.
In another embodiment, the invention discloses a method, wherein the protein is a biotherapeutic.
In yet another embodiment, the invention discloses a method, wherein the protein is a monoclonal antibody or fusion protein.
In another embodiment, the sugars and sugar alcohols present in the sample are glucose, sucrose, trehalose, maltose, sorbitol and mannitol.
In yet another embodiment, the sugars present in the sample are maltose, sucrose and trehalose.
In yet another embodiment, concentration of each sugar or sugar alcohol present in the sample is at least about 10 mg/mL.
In another embodiment, the invention discloses a method, wherein the polymer gel is NH2P-50 gel.
In another embodiment, the invention discloses a method, wherein the diluted sample is centrifuged at 9500xg for 20 minutes.
In another embodiment, the invention discloses a method, wherein the eluent comprises 70% acetonitrile.
In another embodiment, the invention discloses a method, wherein the defined flow rate is 0.3, 0.5. 0.7, 1.0 mL/min, or more preferably 1.0 mL/min.
In another embodiment, the invention discloses a method, wherein the defined column temperature is 300C to 500C, or more preferably 300C.
In an embodiment, the invention discloses a method for determination of one or more of glucose, sucrose, trehalose, maltose, sorbitol and mannitol in a protein sample, wherein the method comprises:
a) diluting the protein sample in water;
b) centrifuging the diluted sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) loading the permeate onto a chromatographic column packed with polyamine bonded polymer gel at a defined column temperature;
d) eluting glucose, sucrose, trehalose, maltose, sorbitol and mannitol bound to the column using an eluent comprising acetonitrile by isocratic elution at a defined flow rate and the defined column temperature;
e) detecting one or more of glucose, sucrose, trehalose, maltose, sorbitol and mannitol using a refractive index detector wherein temperature of the detector is preferably 400C; thereby determining one or more of glucose, sucrose, trehalose, maltose, sorbitol and mannitol present in the protein sample;
wherein concentration of each analyte is at least about 10 mg/mL and
In an embodiment, the invention discloses a method for determination of one or more of maltose, trehalose and sucrose in a protein sample, wherein the method comprises:
a) diluting the protein sample in water;
b) centrifuging the diluted sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) loading the permeate onto a chromatographic column packed with polyamine bonded polymer gel at a defined column temperature;
d) eluting one or more of glucose, sucrose, trehalose, maltose, sorbitol and mannitol bound to the column using an eluent comprising acetonitrile by isocratic elution at a defined flow rate and the defined column temperature;
e) detecting one or more of glucose, sucrose, trehalose, maltose, sorbitol and mannitol using a refractive index detector wherein temperature of the detector is preferably 400C; thereby determining maltose, trehalose and sucrose present in the protein sample;
wherein concentration of each analyte is at least about 10 mg/mL.
Definitions:
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” or “an” as used herein refer to one or more than one (i.e, to at least one) of the grammatical object of the article. The term “at least” is used to indicate that optionally one or more further objects may be present.
The term “biotherapeutic” as used herein refers to a protein-based drug product which is under development for clinical studies for approval or which can be administered to an individual of treatment of a disease.
The term “determining” as used herein includes both qualitative and quantitative measurements. In one aspect of the method, the method of determining as described herein is used to measure the amount of one or more analytes present in the sample.
The expression “one or more” denotes 1 to 10, preferably 1 to 6 or 1 to 5, or 1 to 4, or 1 to 3 or 1 to 2.
The term “sample” as used herein refers to a solution containing a protein-based drug for the purpose of evaluation. The sample may be procured from any stage of the drug development process including downstream processing, pre-formulation process, formulation process, or upto the finished drug product. Typically, the sample is a liquid sample. The sample may contain monoclonal antibodies, fragments thereof or a fusion protein.
The term “standard sugar mixture” refers to a solution containing one or more of 10 mg/mL Maltose monohydrate,Trehalose dihydrate ,Mannitol ,Sorbitol and Sucrose as applicable in each instance.
The term “sugar” as used herein refers to one or more monosaccharides or disaccharides present in the sample. This is exemplified by maltose, trehalose, glucose, mannose, sucrose etc. The term “sugar alcohol” or “polyol” as used herein refers to compounds derived from sugars containing one or more hydroxyl groups. The term is exemplified by mannitol, sorbitol, glycerol, sorbitol etc.
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: Selection of column
A chromatographic separation column is the core of a method that employs HPLC. It is necessary to select the right column that allows separation of all the relevant molecules under analysis. With respect to present invention, the relevant sugars and sugar polyols that are most often part of protein-based drug formulations include glucose, sucrose, trehalose, maltose, sorbitol, and mannitol. The ideal column of present invention can differentiate the above-mentioned analytes as distinct peaks on a chromatogram.
To select the ideal column, two different columns were compared for their respective outcomes: Shodex® EP SC1011-7F column and Shodex®NH2P series,VG-50 column (packed with polyamine bonded NH2P polymer gel). Shodex® EP SC1011-7F column are packed with styrene divinylbenzene copolymer (bearing Ca2+ counterion) and uses a combination of ligand exchange and size exclusion for separation of analytes. On the other hand, Shodex®NH2P series,VG-50 column is packed with polyamine bonded polymer (NH2P) gel.
For the analysis, 10 µL of standard sugar mixture (containing 10 mg/mL Maltose monohydrate,Trehalose dihydrate ,Mannitol ,Sorbitol and Sucrose) was loaded onto each columns. Chromatography method parameters using the respective columns are shown in Table 1 & 2. Sample temperature was maintained at 5±3°C. Analytes were measured using a refractive index detector (RID) which was maintained at a temperature of 400C and data is collected as µRIU (micro-refractive index units) or mV (millivolts). Representative chromatograms showing retention time (RT) vs. millivolts (mV) plot of the standard sugar mixture sample are depicted in Figure 1 & 2. It is clear from the chromatogram output that separation using under the given run and detection conditions gives clear differentiating peaks for sorbitol, mannitol, glucose, sucrose, trehalose, and maltose at the same time.
Parameters Condition/Units
Mobile phase Degassed filtered WFI/ MilliQ water
Column details Shodex® EP SC1011-7F
Column Temperature 61± 2°C
Sample Temperature 5 ± 3°C
Injection Volume 10 µL (for Standards)
Gradient Isocratic
Run Time 30 minutes
Flow Rate 0.8 mL/min
RI Detector temperature 40°C
Table 1: Parameters for method using Shodex® EP SC1011-7F column
Parameters Condition/Units
Mobile phase Acetonitrile (75%)
Column details Shodex®NH2P series,VG-50
Column Temperature 40 ± 2°C as per COA of column (25-50°C)
Sample Temperature 5 ± 3°C
Injection Volume 10 µL (for Standards)
Gradient Isocratic
Run Time 30 minutes
Flow Rate 1 mL/min
Detector temperature 40°C
Table 2: Parameters for method using Shodex®NH2P series,VG-50 column
Example 2: Selection of column temperature
It is important to arrive at the suitable temperature or ranges thereof of the column at which accurate determination of the analyte(s) can be performed as column temperature plays an important role in chromatographic performance. For this, three different column temperatures were considered, viz., 300C, 400C and 500C. 10 µL of standard sugar mixture (containing 10 mg/mL Maltose monohydrate,Trehalose dihydrate, Mannitol ,Sorbitol and Sucrose) was loaded onto the a column maintained at either 300C, 400C and 500C. Samples were run at a flow rate of 1 mL/min for a run time of 30 mins and detected using refractive index (RI) detector at a detector temperature of 400C. Representative chromatograms (retention time (RT) vs. millivolts (mV)) are shown in Figure 3. All the three column temperature conditions tested gave acceptable resolution for all the analytes.
Example 3: Selection of mobile phase
While saccharide separation can be preformed with distilled water as mobile phase, separation of polyols requires a solvent such as acetonitrile in addition. It is important to select the mobile phase conditions that result in the greater peak resolution of that of the analytes under study. For this, two different ratios of acetonitrile: water were selected (75:25 and 70:30). Representative overlay of chromatogram is shown in Figure 4. It was found that retention time of analytes was improved (reduced) without impact on the desired peak profile with the use of 70:30 combination when compared to the former. Hence, mobile phase containing 70% acetonitrile and 30% water was selected for the analysis.
Example 4: Percentage recovery of analyte
Percentage recovery of one of the analytes (sucrose) was checked using the disclosed method. To evaluate this, 10 µL of 5 mg/mL to 60 mg/mL of sucrose standard was injected into the Shodex®NH2P series,VG-50 column and sample was % sucrose recovery was calculated. Output of this study is tabulated in Table 3. The disclosed method was shown to give % recovery within acceptable ranges. (98-101%) for the concentration ranges of standard sucrose solutions analyzed.
Sucrose standard injected
(mg/mL) AUC Back Calculated Conc % Recovery
5 298723 5 98
10 602528 10 98
20 1216010 20 99
30 1865284 30 101
40 2464393 40 100
50 3100359 50 101
60 3645849 59 99
Table 3: Percentage recovery of sucrose in standard sucrose solution
Next, percentage recovery of sucrose in a biopharmaceutical product (DRL-A) was analysed using disclosed method. For this, sample was diluted 5 fold in water and added onto a 30kDa cutoff centrifugal filter. The filter was centrifuged at 9500 x g for 20 minutes. The retentate (containing protein) was discarded and permeate (containing sugars & polyols) was used for further analysis. Analysis conditions are listed in Table 2. Output of this study is tabulated in Table 4. The disclosed method was shown to give % recovery within acceptable ranges. (98-101%) for the concentration ranges of the in-house biopharmaceutical samples analyzed.
Sample I.D AUC dilution fold Expected Conc. (mg/mL) Obseved Conc. (mg/mL) % Recovery
Std sucrose solution 1859402 NA 30 30.3 101.0
DRL_A sample #1 1871638 5 150 152.5 101.7
DRL_A sample #2 2077549 5 150 169.3 112.8
Table 4: Percentage recovery of sucrose in in-house biopharmaceutical sample
Example 5: Interference from non-analytes
Specificity of a method refers to ability the said method to assess the analyte(s) unequivocally in the presence of components which might otherwise be expected to be present in the sample. For example, for evaluating the interference of components in sucrose analysis, which might otherwise be expected to be present in the in-house biopharmaceutical sample (DRL_A), individual components were analyzed individually (eg. Sodium, Phosphate, L-Histidine, D-Mannitol and poloxamer-188) along with the test sample under the same method conditions. Sucrose is expected to elute at RT 9.2 ± 2 minutes. DRL_A sample was processed and analyzed as described in Example 4. Results are shown in Figure 5 as overlay of the output for test sample (DRL_A) and individual components. As is evident from Figure 5, disclosed method was found to be specific and free from interference effects by possible interfering components that may be present in a typical test sample during the analysis time and conditions performed.
,CLAIMS:We claim:
1. A method for determination of one or more of sugar analyte or sugar alcohol analyte present in a protein sample, wherein the method comprises:
a) diluting the sample in water;
b) centrifuging the diluted sample using a 30 kDa cut off centrifugal filter and collecting the permeate;
c) loading the permeate onto a chromatographic column packed with polyamine bonded polymer gel at a defined column temperature;
d) eluting one or more of the analyte bound to the column using an eluent comprising acetonitrile by isocratic elution at a defined flow rate and the defined column temperature;
e) detecting one or more of the analyte using a refractive index detector wherein temperature of the detector is preferably at 400C;
f) determining one or more of the analyte present in the protein sample by comparing output with reference to that of a standard mixture;
wherein the analyte determined is one or more of glucose, sucrose, trehalose, maltose, sorbitol or mannitol; and
wherein concentration of each analyte is at least about 10 mg/mL.
2. The method as claimed in claim 1 wherein, the sample temperature is 50C, 60C, 70C, 80C, or more preferably 50C.
3. The method as claimed in claim 1 wherein, the sample is diluted 2 fold, 3 fold, 4 fold, 5 fold, or more preferably 5 fold.
4. The method as claimed in claim 1 wherein, wherein the protein is a monoclonal antibody or fusion protein.
5. The method as claimed in claim 1 wherein, the polymer gel is NH2P-50 gel.
6. The method as claimed in claim 1 wherein, the defined column temperature is 300C to 500C, or more preferably 300C.