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 a biologic drug product sample that is crystalline in nature, using oven method of Karl Fischer titration wherein the method allows moisture content determination with high level of accuracy, specificity, linearity, precision and repeatability.
DETAILED DESCRIPTION OF DRAWINGS
Figure 1: Graphical plot illustrating sample exposure time vs % moisture content
Figure 2: Graphical plot showing observed % moisture content at condition of 90°C, 60 mL/min
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 crystalline 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) transferring a defined amount of sample into a vial;
b) hermetically sealing the vial;
c) positioning the vial in an oven that facilitates uniform sample heating;
d) puncturing the seal to insert tubing that allows passage of a carrier gas devoid of extrinsic moisture through the sample;
e) passing the carrier gas at a flow rate of about 30 to 120 mL/min through the sample;
f) heating the sample to about 900C; and
g) measuring the moisture content in the sample electrometrically by Karl Fischer titration method;
wherein, the sample is intact before transferring into the vial;
and wherein the method adequately excludes extrinsic moisture content.
In a further embodiment, the Karl Fischer titration method is a coulometric titration method.
In yet another embodiment, the carrier gas flow rate is preferably 30 mL/min, 60 mL/min or 120mL/min; more preferably 60 mL/min or 120mL/min; most preferably 60 mL/min.
In another embodiment, the sample in the vial weighs at least 0.1 g.
In another embodiment, the sample in the vial weighs at least 0.5 g.
In any of the preceding embodiments, the lyophilized drug sample is a crystalline lyophilized drug sample.
In any of the preceding embodiments, the lyophilized drug sample is a biologic product.
In any of the preceding embodiments, the lyophilized drug is CTLA4-Ig fusion protein.
In any of the preceding embodiments, the carrier gas is dry air.
Definitions:
The term “adequately excludes extrinsic moisture” as used herein indicates that the method is able to pre-empt increase in % extrinsic moisture content in the sample by at least about 1-24%.
The term “biologic product” 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 “extrinsic moisture” refers to moisture content excluding that from the sample to be analyzed (e.g, atmospheric moisture).
The term “intact” means that the sample is not broken or subjected to pulverization.
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.
The term “sample handling” as used herein refers to the time period of exposure of the sample to atmosphere before transferring the same into vial. The purpose of exposure may be for weighing or crushing of cake 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.
Examples of present invention describe residual moisture content determination in, but not limited to, CTLA4-Ig fusion protein sample that is crystalline in nature and dry air as carrier gas.
Example 1: Carrier gas flow rate
To determine carrier gas flow rate, 0.1 g lyophilized drug cake was weighed, crushed with a clean, dry spatula into a fine powder and immediately transferred into a sample vial (in no more than 5 mins time). Vial was hermetically sealed and analyzed for residual moisture content by Karl Fischer titration. For this, sample was heated at an oven temperature of 800C and at different carrier gas flow rates.
The experimental conditions and results are summarized in Table 1.
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 sample weight (in grams)

Sample No. Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time of analysis (min) Comments
1 80 120 1.5 23.0 Titration was completed
2 80 60 1.5 27.8 Titration was completed
3 80 30 1.4 32.3 Titration was completed
Table 1
No significant difference was observed in the recorded % moisture content values between the samples analyzed at different flow rates (30, 60 & 120 mL/min). Understandably, at lower flow rates, the time taken to determine the % moisture content increased with no significant change in observed moisture content. Visibly, there was no charring of sample at 800C.
Example 2: Oven temperature
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 those that result in near complete moisture content release with no product charring during analysis by the oven method.
To determine oven temperature, 0.1 g lyophilized drug cake was crushed with a clean, dry spatula into a fine powder and immediately transferred into a sample vial (in no more than 5 mins time). Vial was hermetically sealed and analyzed for % residual moisture content by Karl Fischer titration coulometrically. For this, sample was heated at different oven temperatures (800C, 1000C & 1200C) at carrier gas flow rate of 60 mL/min. Moisture content was determined as explained in Example 1.
The experimental conditions and results are summarized in Table 2.

Sample No. Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time of analysis (min) Comments
1 120 60 2.2 36.4 Complete moisture release, but cake charred
2 100 60 2.7 23.0 Complete moisture release
3 80 60 1.5 27.8 Incomplete moisture release
Table 2
Sample heated to 1200C was found to show visible charring of cake. In contrast, sample heated to 800C resulted in low % moisture content, indicating incomplete moisture release. At 1000C, moisture content release was observed without charring of cake.
Example 3: Sample handling
It is important to understand the impact of environmental moisture on the cake during sample handling (e.g., during opening of vial). In order to study this, the cake was exposed to outside environment for different time intervals and the moisture content was analyzed at oven temperature of 1000C and a carrier gas flow rate of 120 mL/min. The experimental conditions and results are summarized in Table 3.

Sample No. Exposure time (min) Moisture content (%) Time of analysis (min) Additional moisture absorbed in comparison to sample exposure for 0 min (%) Comments
1 0 1.27 13.1 0 Sample stoppered immediately (T0)
2 5 1.58 14.6 24.4 Sample stoppered after 5 mins (t5)
3 10 2.07 11.3 62.9 I Sample stoppered after 10 mins (T10)
4 15 2.72 18.9 114.2 Sample stoppered after 15 mins (T15)
Table 3
Exposure of product to environment for 15 min resulted in almost doubling of recorded moisture content (1.27% at T0 to 2.72% at T15).
Example 4: Intact cake analysis
As a proportional increase in residual moisture was observed with increasing exposure to atmospheric moisture, further experiments were performed using whole intact cake (to minimize exposure ) of different weights (0.1/0.5 g) at a flow rate of 60 & 120 mL/min. Results of experiment using 0.5 g cake are tabulated in Table 4.

Sample No. Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time of analysis (min) Comments
1 120 120 NA NA Titration incomplete. Cake was charred
2 100 120 2.04 100.7 Titration incomplete. Cake was charred
3 90 120 1.3 85.0 Titration was completed. No charring was observed
4 90 60 1.2 87.5 Titration was completed. No charring was observed
Table 4
It was found that samples (of 0.5 g weight) analyzed at 100°C and 120°C displayed charring of cake (indicating decomposition). Whereas, sample analyzed at 90°C did not char and in which case titration was also found completed. In the same way, samples analyzed at 90°C, with varying flow rates i.e. 60mL/min and 120mL/min showed similar moisture content release with no major difference in the sample analysis time.
Next, to check for repeatability and reproducibility in 0.1 g sample cake, three samples of 0.1 g weight each were analyzed at 90°C oven temperature and 60mL/min flow rate. To eliminate vial to vial variation in moisture content due to sample handling, 0.1 g sample was aliquoted from a single lyophilized vial into three separate vials and immediately crimped and subject to moisture content estimation. Experimental conditions and results are tabulated in Table 5 and Figure 2.

Sample ID Temperature
(°C) Flow rate (mL/min) Moisture content (%) Time (min) Comments
1 90 60 1.51 25.4 Titration was complete
2 90 60 1.58 30.6 Titration was complete
3 90 60 1.50 25.5 Titration was complete
Average 1.53
SD 0.0436
RSD 2.849

Table 5
All three samples analyzed showed no major difference in % moisture content values, indicating repeatability and reproducibility of analysis in a smaller sample size.
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) transferring a defined amount of sample into a vial;
b) hermetically sealing the vial;
c) positioning the vial in an oven that facilitates uniform sample heating;
d) puncturing the seal to insert tubing that allows passage of a carrier gas devoid of extrinsic moisture through the sample;
e) passing the carrier gas at a flow rate of about 30 to 120 mL/min through the sample;
f) heating the sample to about 900C; and
g) measuring the moisture content in the sample electrometrically by Karl Fischer titration method;
wherein, the sample is intact before transferring into the vial;
and wherein the method adequately excludes extrinsic moisture content.
2. The method as claimed in claim 1 wherein, the Karl Fischer titration method is a coulometric titration method.
3. The method as claimed in claim 1 wherein, the carrier gas flow rate is preferably 30 mL/min, 60 mL/min or 120mL/min; more preferably 60 mL/min or 120mL/min; most preferably 60 mL/min.
4. The method as claimed in claim 1 wherein, the sample in the vial weighs at least 0.1 g.
5. The method as claimed in claim 1 wherein, the sample in the vial weighs at least 0.5 g.
6. The method as claimed in claim 1 wherein, the lyophilized drug sample is a crystalline lyophilized drug sample.
7. The method as claimed in claim 1 wherein, the lyophilized drug sample is a biological product.
8. The method as claimed in claim 1 wherein, the lyophilized drug is CTLA4-Ig fusion protein.
9. The method as claimed in claim 1 wherein, the carrier gas is dry air.