DESC:FIELD OF THE INVENTION
The present invention relates to a method for determining moisture content in a lyophilized drug product.
BACKGROUND OF THE INVENTION
‘Residual moisture content’ or ‘moisture content’ of a lyophilized drug product when stored in the dried state, strongly affects its quality, stability and shelf life and can also be a limiting factor in the accuracy calculations of the drug content. In the case of a protein based biological drug product, while “dry” is a state of having an acceptable level of residual moisture, “over-drying” is a state at which the stability of the protein is at stake. Hence, during formulation development, a careful balance in the residual moisture content has to be achieved between drying ‘sufficiently’, taking care not over-dry to the detriment of protein stability.
For this reason, determining residual moisture content is one of the important critical quality attributes mandated by regulatory bodies. By far, a widely accepted and proven technique for moisture content determination is the Karl Fischer Titration (KFT) method wherein, titration is performed by volumetric or coulometric means.
Karl Fischer (KF) Titration is based on the Bunsen reaction wherein, SO2 is oxidized by I2, in the presence of water (from the sample). In this reaction, water and iodine are consumed in a 1:1 mole ratio. Once the reaction consumes all the water present in the sample, excess iodine is detected by the electrode, marking the end point of titration. In the coulometric method (the preferred method for measuring moisture content in lyophilized drugs), iodine is generated electrochemically in situ during the titration and water is quantified based of the total charge passed (Q), as measured by current (A; amperes) and time (s; seconds), according to the following relationship:
Q = 1 C (Coulomb) = 1 A x 1 s where, 1 mg H2O corresponds to 10.72 C.

In cases where the rate of release of water/moisture from the sample is slow, or when there could be a risk of side reactions from other components in the sample, the sample can be heated (using an oven) and a carrier gas can be used to transfer the released moisture to the titration cell to speed up the reaction and to minimize false positives/negatives. This is referred to as the ‘oven method’ for sample preparation of KF titration. However, over-heating can cause decomposition of the sample and/or heat labile excipients that can impact test result. Obviously, under-heating can lead to incomplete moisture release. In addition, the amorphous or crystalline nature of the sample itself can influence the choice of the process parameters in a major way.
Also, flow rate of the carrier gas is a parameter that can impact rate of transfer of released water and time of attainment of the reaction end point. Additionally, utmost care should be taken to minimize exposure of the sample to extrinsic moisture during sample handling, ensure uniform and maximal extraction/release of moisture into the carrier gas etc. Much of this is dependent upon the nature and type of sample to be tested.
As moisture content plays a key role in solid-state stability of protein-based lyophilized biological drug formulations, there is a need to develop a method for accurate and precise moisture content determination, with the right parameters, taking into consideration the nature of the sample.
SUMMARY OF THE INVENTION
Accordingly, present invention provides a method for accurate determination of residual moisture content in an amorphous biologic drug product sample that is amorphous in nature, using oven method of Karl Fischer titration wherein the method allows moisture content determination with acceptable levels of accuracy, specificity, linearity, precision and repeatability.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses an efficient method for measuring moisture content in a drug product. More particularly, present invention discloses an efficient method for measuring moisture content in a biologic drug product that is amorphous in nature.
The present invention discloses a method for measurement of residual moisture content in a biologic drug product sample using oven method of Karl Fischer titration.
In an embodiment, the invention discloses a method for measuring residual moisture content in a lyophilized drug sample, the method comprising:
a) weighing a defined amount of sample in a vial;
b) hermetically sealing the vial;
c) loosening the sample in vial by gentle tapping;
d) positioning the vial in an oven that facilitates uniform sample heating;
e) puncturing the seal to allow passage of a carrier gas devoid of extrinsic moisture through the sample;
f) passing the carrier gas at a flow rate of about 60 mL/min to 80 mL/min through the sample;
g) heating the sample to about 1000C to 1200C; and
h) measuring the moisture content in the sample electrometrically by Karl Fischer titration method with a pause time of at least 10 minutes;
wherein, the method reduces interference by extrinsic moisture and can accurately measure the residual moisture content in the sample.
In yet another embodiment, the sample is heated to about 1000C, 1010C, 1020C, 1030C, 1040C, 1050C, 1060C, 1070C, 1080C, 1090C, 1100C, 1110C, 1120C, 1130C, 1140C, 1150C, 1160C, 1170C, 1180C, 1190C or 1200C.
In yet another embodiment, the sample in the vial weighs about 0.1 g to about 0.5 g.
In another embodiment, the method is able to determine the residual moisture content with the accuracy range of 75% to 91%.
In any of the preceding embodiments, the lyophilized drug sample is amorphous in nature.
In any of the preceding embodiments, the lyophilized drug sample is a biologic product.
In any of the preceding embodiments, the carrier gas is dry air.
In any of the preceding embodiments, the Karl Fischer titration method is a coulometric titration method.
Definitions:
The term “accuracy” of the method refers to closeness of agreement between a value which is accepted either as a conventional true value or as an accepted reference value and the value measured.
The term “biologic” as used herein refers to a diagnostic, preventive, or therapeutic preparation derived from a biological source.
The term “carrier gas” refers to the medium devoid of extrinsic moisture that carries moisture from the sample to the titration cell.
The term “end point of titration” refers to the point when the potential at the detector electrode of the Karl Fischer titration unit drops below a specified predefined value. The predefined value of present invention is 50.0 mV.
The term “extrinsic moisture” refers to moisture content excluding that from the sample to be analyzed (e.g, atmospheric moisture).
The term “pause time” refers to the time period for which current flow through the generator electrode is withheld between start of sample heating and start of analysis.
The term “residual moisture content” or “moisture content” in a drug sample refers to the water content that is present in the sample substance and that can be expelled (for example, by heating) without essentially altering the chemical composition of the substance
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.
Examples of present invention describe residual moisture content determination in, but not limited to, anti-a4ß7 antibody sample, that is amorphous in nature.
Example 1: Oven temperature, flow rate and pause time
The 860 KF Thermoprep that allows a heating temperature range from 50°C to 250°C was used for thermal sample preparation. Various temperatures were screened so as to arrive at temperature/temperature ranges that would result in near complete moisture content release and that also ensures no visible product charring.
A defined weight of sample was carefully transferred into a sample vial, which was then hermetically sealed and analyzed for residual moisture content by Karl Fischer titration by heating at different oven temperatures and at differing flow rates of the carrier gas. The percentage moisture content (‘% moisture’) was calculated based on the formula:
% moisture = A- blank value
M X 10000
wherein,
A is moisture content (in µg)
blank value is the average moisture content (in µg) from the blank vial.
M is the weight of the sample (in g)
Moisture content was also measured by varying the pause time. The experimental conditions and results are summarized in Table 1.

Sample No. Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time of analysis (min) Comments
1
150 100 NA NA Run stopped as it took more than 1 hr.; Cake charred
2 120 100 NA NA Run stopped as it took more than 1 hr.; Cake charred
3 120 80 1.04 58:34 Pause time of 10 min
4 120 60 0.82 53:43 Pause time of 10 min
5 100 80 0.84 56:51 Pause time of 10 min
6 100 60 0.89 58:39 Pause time of 10 min
Table 1
It was found that sample heated to a temperature above 1000C and flow rate below 80 mL/minute releases moisture without any visible charring of cake with end point reached within an hour of start of study. Further, introducing a pause time (time period for which current flow through the generator electrode is withheld after sample heating and start of analysis) of 10 minutes in the analysis yielded superior results.
Example 2: Sample handling
Experiments to minimize sample analysis time was performed. For this, sample in vial was loosened by gentle tapping before analysis. Experiment was carried out at sample heating of 1200C, flow rate at 80 mL/min or 60 mL/min. Results are tabulated in Table 2.

Sl. No. Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time of analysis (min) Sample loosened by tapping Pause time
1 120 80 1.04 58:34 X 10 min
2 120 60 0.82 53:43 X 10 min
3 120 60 0.81 28:28 ? 10 min
4 120 80 0.76 28:40 ? 10 min
5 120 60 0.93 43 ? 5 min
Table 2
No significant difference in moisture content values were observed between the experiments where flow rate of 60ml/min and 80ml/min was applied. In samples loosened by tapping prior to analysis, time taken to determine the moisture content was considerably reduced with no significant change in the observed moisture content.
Example 3: Method qualification for specificity, linearity, precision and accuracy
Specificity of the method refers to the ability to assess unequivocally the moisture content in the presence of other components present in the sample. Typically these might include excipients, impurities etc. Linearity of the assessment refers to the lowest and highest levels at which an analyte can be measured accurately.
The above aspects were determined by injecting a range of known amount of water standards (0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 g). Results are summarized in Table 3.
A linear positive response was observed upon analyzing standards while, blank measurement displayed negligible response. R2 value (coefficient of determination) was observed to be greater than 0.99 with the 7 standard points.

Points Water standard (g) Observed moisture content (µg) Observed moisture content (%)
1 Blank (0) 222.1 0.00
2 0.10 5287.5 0.51
3 0.25 12891.0 1.27
4 0.50 25481.1 2.53
5 0.75 38023.3 3.78
6 1.00 50440.6 5.02
7 1.25 62630.6 6.24
8 1.50 75219.6 7.50
Table 3

Further, precision of the method (closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions) was measured by assessing intra-assay repeatability and intermediate precision tests. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability was assessed by injecting six replicates from the same batch. The results are summarized in Table 4. The relative standard deviation was found to be 2.2-2.4 %, indicating the level of precision of the data.
Sample Details Moisture content (µg) Moisture content (%)
1 4330.57 0.87
2 4496.87 0.90
3 4607.67 0.92
4 4505.27 0.90
5 4516.47 0.90
6 4644.67 0.93
Mean (n=6) 4516.92 0.90
SD 109.38 0.02
RSD 2.42 2.22
Table 4
Further, intermediate precision of the method was assessed by performing the method on two different days (inter-day precision) by two different analysts (inter-analyst precision). Results are summarized in Table 5 & Table 6. The RSD for the samples analyzed was found to be < 3.4%.
Day-1 / Analyst-1 Day-2 / Analyst-2
Sample No. Moisture content (µg) Moisture content (%) Sample Details Moisture content (µg) Moisture content (%)
1 4330.57 0.87 1 4339.44 0.87
2 4496.87 0.9 2 4602.44 0.92
3 4607.67 0.92 3 4697.64 0.94
4 4505.27 0.9 4 4588.04 0.92
5 4516.47 0.9 5 4611.24 0.92
6 4644.67 0.93 - - -
Average (n=6) 4516.92 0.90 Average (n=5) 4567.76 0.91
SD 109.38 0.02 SD 134.63 0.03
RSD 2.42 2.22 RSD 2.95 3.29
Table 5
Combined results (n=11)
Average moisture content (µg) 4540.03
SD 118.06
RSD 2.6
Table 6
Accuracy of the method indicates the closeness of agreement between a value which is accepted either as a conventional true value or as an accepted reference value and the value measured. Accuracy for moisture content was performed by spiking of water standard over a range of 50% (0.1g standard) and 150% (0.4g standard) concentrations in the sample mentioned in repeatability experiment. Theoretical moisture content in the spiked samples was calculated and is presented in Table 7. Accuracy of the spiked samples with water standards was observed in the range of 75.2% to 91.4% which is well within the acceptable accuracy range of 70% to 130%. Thus, the method is specific, linear, precise and accurate.

Sample ID Observed moisture content (µg) Observed moisture content (%) Expected moisture content (%) Percentage
Recovery (%) Average recovery (%)
Sample – 0.1g 7674.29 1.28 1.4 91.4 89
7320.89 1.22 87.2
7424.99 1.24 88.4
Sample – 0.4g 21313.8 2.37 2.9 81.7 80.6
19620.7 2.18 75.2
22190.3 2.47 85
Table 7
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 residual moisture content in a lyophilized drug sample, the method comprising:
a) weighing a defined amount of sample in a vial;
b) hermetically sealing the vial;
c) loosening the sample in the vial by gentle tapping;
d) positioning the vial in an oven that facilitates uniform sample heating;
e) puncturing the seal to allow passage of a carrier gas devoid of extrinsic moisture through the sample;
f) passing the carrier gas at a flow rate of about 60 mL/min to 80 mL/min through the sample;
g) heating the sample to about 1000C to 1200C; and
h) measuring the moisture content in the sample electrometrically by Karl Fischer titration method with a pause time of at least 10 minutes;
wherein, the method reduces interference by extrinsic moisture and can accurately measure the residual moisture content in the sample.
2. The method as claimed in claim 1 wherein, the sample is heated to about 1000C, 1010C, 1020C, 1030C, 1040C, 1050C, 1060C, 1070C, 1080C, 1090C, 1100C, 1110C, 1120C, 1130C, 1140C, 1150C, 1160C, 1170C, 1180C, 1190C or 1200C.
3. The method as claimed in claim 1 wherein, the sample in the vial weighs about 0.1 g to about 0.5 g.
4. The method as claimed in claim 1 wherein, the method is able to determine the residual moisture content with the accuracy range of 75% to 91%.
5. The method as claimed in claim 1 wherein, the lyophilized drug sample is amorphous in nature.
6. The method as claimed in claim 1 wherein, the lyophilized drug sample is a biologic product.
7. The method as claimed in claim 1 wherein, the carrier gas is dry air.
8. The method as claimed in claim 1 wherein, the Karl Fischer titration method is a coulometric titration method.