Journal of Pharmaceutical Sciences & Emerging Drugs ISSN: 2380-9477

Research Article, J Pharm Sci Emerg Drugs Vol: 4 Issue: 2

Formulation Characterization of Parathyroid Hormone PTH (1-34)Coated on a Novel Transdermal Microprojection Delivery System:Water Determination

Mahmoud Ameri1*, Shelley C Fan1 and Yuh Fun Maa2
1Zosano Pharma, Inc., 34790 Ardentech Court, Fremont, CA 94555, USA
2Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
Corresponding author : Mahmoud Ameri
Zosano Pharma, Inc., 34790 Ardentech Court, Fremont, CA 94555, USA
[email protected]
Received: October 25, 2016 Accepted: November 07, 2016 Published: November 15, 2016
Citation: Ameri M, Fan SC, Maa YF (2016) Formulation Characterization of Parathyroid Hormone PTH (1-34) Coated on a Novel Transdermal Microprojection Delivery System: Water Determination. J Pharm Sci Emerg Drugs 4:2. doi:10.4172/2380-9477.1000115


This study assessed the feasibility of determining the water content of the PTH (1-34) formulation coated on a novel transdermal microprojection delivery system, ZP-PTH. Thermogravimetric analysis (TGA) and Karl Fischer (KF) titration were used for water analysis.Headspace moisture was determined by internal vapor analysis and a hand-held moisture sensor. The moisture sorption isotherm was established by moisture sorption analyzer. PTH (1-34) API and formulation showed several weight-loss phases in TGA thermograms. Water loss is not a single-phase event, occurring over a wide temperature range. The typical water amount in a single ZP-PTH coated array is below the sensitivity of TGA and KF titration. Combining 20 coated array samples together in a single extract made KF analysis is possible but impractical due to strenuous sample preparation requirements and controls. The moisture sorption isotherm allowed accurate prediction of water content when the headspace moisture level is known. We demonstrated that TGA and KF titration are not practically possible for determining the water content in ZP-PTH. Simple alternative approaches can ensure that the desired water content in the ZP-PTH coating is achieved without direct water measurement. This study provides scientific rationales to justify the application of unconventional approaches.

Keywords: Microneedles; Parathyroid hormone; Transdermal microprojection delivery System; Drug-coating; Water Content; TGA; Karl Fischer titration; Moisturesorption isotherm


Microneedles; Parathyroid hormone; Transdermal microprojection delivery System; Drug-coating; Water Content; TGA; Karl Fischer titration; Moisture sorption isotherm


To ensure pharmaceutical product quality and consistency, the drug product needs to be thoroughly characterized during development, which eventually supports setting rational specifications for clinical and commercial manufacturing. International Conference on Harmonization (ICH) provides a guideline on setting specifications to important attributes of drug products of common dosage forms such as solid oral drug products, liquid oral drug products, and parenterals and encourages the application of this guideline to other dosage forms [1]. This generalization can become the underlying thinking of the regulatory agent even in assessing the proposed specifications of a drug product originated from a novel technology or a unique dosage form. Thus, it is the innovator’s burden to find a creative way to characterize the product or propose that alternative methods are more suitable for the intended characterization. This investigation demonstrated such challenges in determining the water content of a dry parathyroid hormone 1-34, or PTH(1-34), formulation coated on a novel transdermal micro projection delivery system, or ZP-PTH [2-4].
ZP-PTH features a small drug-coated patch (5 cm2 in area) seated on a patch retainer ring. The patch is applied to the skin with a handheld reusable applicator (Figure 1a). The patch consists of a titanium microprojection array (~1,300 micro projections per 2 cm2 and 190 μm in length in Figure 1b) attached to the center of an adhesive backing. Drug formulation is coated on the tip of each microprojection. When the patch is applied onto the skin, the drug-coated microprojections penetrate through the superficial skin barrier layer into the epidermal/ dermal layers (50-150 micro meters in depth), where the drug formulation rapidly dissolves in the interstitial fluid and releases into the skin for microcapillary uptake and systemic absorption. In a Phase 2 dose-finding clinical study, the 40 mcg dose ZP-PTH (i.e., 80 mcg total formulations solid) was found to be most effective in building bone mineral density, the primary pharmacodynamics end point [5]. ZP-PTH is currently entering a Phase 3 human trial.
Figure 1: Transdermal microprojection patch delivery system – (a) applicator; (b) drug-coated patch; (c) microprojection array.
The most unique feature of the product is the uniform distribution of a minute amount of PTH (1-34) formulation (0.03 mcg PTH or 0.06 mcg of total formulation solid) on the tip of each microprojection using a novel dip-coating technology [4]. The microprojection tip features a small arrow-head (Figure 1c) with a dimension of 100 μm in height, 115 μm in width (the surface area of ~5.75×10-5 cm2), and 25 μm in thickness. Due to this special product configuration, this solid-state formulation is unique to all other dosage forms. Although a significant amount of development work on relevant microneedle technologies has been published [6-9], product development of these technologies is still in an early stage, preclinical or Phase 1 clinical testing. Therefore, their approaches to product characterization have not yet been disclosed.
Water is a potent plasticizer for amorphous glasses because of its low Tg (-135ºC) [10]. Water content determination is normally required for solid-state, particularly lyophilized, biopharmaceutical powder formulations due to the water’s destabilizing effect on proteins/peptides by lowering the formulation’s Tg [11,12]. For the same reason, the FDA required the water content of ZP-PTH drug formulation be measured and be part of finished product release specifications despite the major difference of the two dosage forms. The standard powder dosage form can be conveniently weighed out and loaded into the sample cell/pan, i.e., easy sample weight adjustment to exceed the detection limit of the method. However, there is no such flexibility for ZP-PTH where 80 mcg of formulation is uniformly loaded on a titanium array whose weight is ~15 mg. Thus, the weight of the formulation makes up ~0.5% of the weight of the coated array and the water content may be ~0.005% of the total sample weight. Certainly, the three-dimensional array configuration (a circular area of 2 cm2) makes sample loading difficult, if not impossible. In addition, the exceedingly large surface area of the array that is not coated by formulation may allow moisture absorption during sample preparation, thereby compromising the accuracy of the test. Overall, identifying a water determination method for the ZP-PTH formulation is a challenge. Still, the strategy here is to evaluate water determination methodologies commonly used in pharmaceutical applications for the purpose of gaining easy access to commercially available instrumentation, avoiding the lengthy development time required, and minimizing method validation hurdles for new methodologies.
Loss-on-drying and titration may be the most commonly used methodologies listed in the US Pharmacopeia (USP) for water content determination [9]. To analyze samples of low water contents, thermogravimetric analysis (TGA) is the most sensitive loss-ondrying method [13,14] while Karl Fischer (KF) is the most effective titration approach [15]. Another frequently used method is near infrared (NIR) spectroscopy [16]. However, this method has its drawbacks for ZP-PTH. It requires calibration samples of different water contents to be determined by a direct method such as TGA or KF titration. The sensitivity of NIR spectroscopy is lower than TGA and Karl Fischer and requires a water amount in the milligram range. Therefore, this study focused on evaluating the feasibility of TGA and Karl Fischer for determining the water content in ZP-PTH.

Experimental Methods

Synthetic PTH (1-34) acetate (Lot #FPTH0501) was supplied by Bachem Americas (Torrance, CA) with an initial purity of 99.5%, water content of 9.0%, and acetate content of 5.1%. Sucrose NF (High Purity Low Endotoxin Grade) was obtained from Pfanstiehl Laboratories (Waukegan, IL). Polysorbate 20 (Crillet 1 HP, high purity, low peroxide) was sourced from Croda (Edison, NJ). HCl and EDTA, all USP Grade, were sourced from Sigma Chemical Company (St. Louis, MO). Titanium metal sheet (Commercially pure Grade 2, 25 μm in thickness) was obtained from Hamilton Precision Metals (Lancaster, PA).
The ZP-PTH system consists of a 2 cm2 titanium array of 1,300 microprojections with a length of 190 μm (Kemac, Azusa, CA). The length and the width of the microprojection head is 100 μm and 115 μm, respectively, with a tip angle of 60º (Figure 2). Other patch components include polycarbonate ring (Jatco, Union City, CA), adhesive patch (Medical Tape 1523, 3M, St. Paul, MN), 3 Å molecular sieve desiccant sachet (3.5 g Minipax, Multisorb, Buffalo, NY), and an aluminium foil pouch (Mangar, New Britain, PA). This study also involved a different form of 3 Å molecular sieve desiccant, 1.0 gram of which was co-molded into the polycarbonate ring using polyethylene glycol as the channelling agent. This co-molded desiccant ring was supplied by CSP Technologies (Auburn, AL).
Figure 2: TGA thermograms, % weight lose and derivative weight (°C/%), for PTH(1-34) API heated at 10 °C/min (a) and 20 °C/min (b).
Microprojection arrays, coating, and packaging: Titanium microprojection arrays were fabricated by a photo-chemical etching and formed using a controlled manufacturing process [17].
Coating was conducted at ambient temperature utilizing a roller drum, rotating at 50 rpm, in a drug formulation reservoir (2 mL in volume controlled at 1ºC) to produce a thin film of controlled thickness of ~100 μm in thickness [4,18,19]. Microprojection arrays are dipped into the thin film, and the amount of coating is controlled by the number of dips (passes) through the drug film. The time between each dip is approximately 5 seconds which is sufficient to dry the coated liquid formulation under the ambient condition.
After coating, the coated array was assembled with adhesive and retainer ring into the drug coated patch (Figure 1b). The patch was then nitrogen purged and heat sealed in a Mangar foil pouch containing a 3 Å molecular sieve desiccant sachet. The sachet is not needed if a co-molded desiccant ring is used.
PTH(1-34) content measurement: PTH(1-34) coated array samples were extracted by purified water. PTH(1-34) concentration was measured by UV-Vis spectroscopy (Model 8453, Agilent Technologies, Santa Clara, CA) using 280 nm as the detection wavelength and 360 nm as background correction and the concentration was calculated against a standard curve.
Reverse-phase HPLC (RP-HPLC) for acetate quantification: RP-HPLC was used to quantify acetate in the PTH (1-34) drug formulation. The acetate in the formulation was fully protonated with phosphoric acid and then separated from PTH (1-34) using a Luna C18(2) column (4.6 mm ID × 250 mm, 5 μm) (Phenomenex CA, USA) at ambient temperature. The eluted acetate was detected by UV absorption at 210 nm. Elution was isocratic at a flow rate of 1.3 mL/ minute using a mobile phase composed of a mixture of ammonium hydroxide (0.14%) (Product# 338818, Sigma-Aldrich Corp., MO, USA) and phosphoric acid (0.60%) (Fluka Product #79606, Sigma- Aldrich, MO, USA) with 2% methanol (JT Baker Product #9093, Mallinckrodt Baker, NJ, USA) for the separation segment, followed by a stepped washing segment using the same buffer with 70% methanol.
Chromatography was performed using an HPLC system (1100 series, Agilent Technologies, Inc., CA, USA) provided with a binary pump, a thermostatted autosampler, a thermostatted column compartment, and a multiple wavelength DAD/UV detector. Data were collected and analyzed using a Turbochrom Client Server Software, version 6.2 (Perkin Elmer, Inc).
Moisture sorption analysis: The moisture sorption/desorption profile of the solid sample was established using a vapor sorption analyzer (SGA-100, VTI Corporation, Hialeah, FL) over a range 5-60% relative humidity (RH) at 10% RH intervals. Equilibrium criteria used for analysis was less than 0.01% weight change in 2 hours, with a maximum equilibration time of 3 hours if the weight criterion was not met.
Establishing equilibration conditions: The PTH (1-34) formulation, lyophilized or coated on arrays, were equilibrated in an environmental condition of different percent relative humidity (%RH). In desiccators, a RH of ≤ 1.4% could be achieved using 3A molecular sieves desiccant sachets. Saturated salt solutions of NaOH and K2CO3 were used to create the conditions of 10% RH and 47% RH, respectively. For establishing a 60% RH environment, an automated humidity controlled chamber (Model 6010, Caron, Marietta, OH) was used.
Thermogravimetric Analysis (TGA): The powder sample or the coated array was loaded into a sample pan of TGA instrument (Module Q500, TA Instrument, New Castle, DE) after the system’s microbalance was tared. The system recorded the initial weight and began to ramp up the oven temperature at the rate of 10 or 20ºC/min. Percent weight loss was recorded as a function of temperature.
Karl Fischer (KF) titration: Moisture content was measured by the KF coulometric method using a Metrohm model #756 titration cell (Brinkmann, Westbury, NY). To avoid ambient air which would induce moisture adsorption by the formulation during sample preparation, the KF apparatus was placed inside a nitrogen-purged glove box (Model 830-ABC, Plas-Labs, Lansing MI) with a relative humidity of <10%. KF reagent was treated with molecular sieve desiccant prior to use.
Sample preparation: Each KF sample represents a coated or uncoated titanium array (Figure 1). Since the instrument manufacturer recommends each sample containing at least 50 mcg of water for accurate measurement, each KF measurement may require combining multiple samples depending on the water content in each sample. Due to small sample size and the formulation’s sensitivity to the surrounding moisture, cautious procedures were taken for sample preparation. All coated array samples were carefully stacked in a glass vial and equilibrated in a controlled environment with a %RH similar to the environment each sample had been exposed to. After equilibration, each glass vial was crimped and sealed before titration. Additionally, an equal number of control samples (non-coated arrays) were prepared following the same procedure. The formulation’s water content was obtained by subtracting the water content of the controls from that of the samples. In the case of lyophilized powdered samples, empty vials were used as control.
KF titration: A known volume of pre-dried KF solvent was injected into sample vials using an airtight syringe (Model 1750, Hamilton, Reno, NV) for formulation extraction. After extraction, a known volume of extraction solution was drawn from the vial and injected into the KF titration cell for water content analysis. The total water content for each measurement is adjusted by the ratio of volume used for extraction and volume applied to the titration cell. Triplicate measurements were made for each condition. The final water amount measured was corrected (subtracted) by water measured from the control samples (average of three).
Headspace analysis (Internal vapor analysis): A headspace gas sample was taken and subjected to quadrupole mass spectroscopy analysis for the identification and quantitation of low molecular weight volatile compounds, including moisture, oxygen, nitrogen, and volatile organics. Briefly, the pouch sample was placed in a test chamber sealed against a Viton™ O-ring. A pin pierced the pouch through the center of the O-ring to take a headspace sample, which was then subjected to a mass spectrometer (Model IVA-110, Oneida Research Services, Whitesboro, NY).
Headspace analysis (Data tracer for monitoring moisture in sealed foil pouch): The data tracer (Datatrace II, serial numbers: M3H22492, M3H22487) was obtained from Mesa Labs (Lakewood, Colorado).
The data tracer (probe) was programmed per manufacturer’s instructions and then placed in the pouch. After monitoring, moisture (%RH) data as a function of time was retrieved from the data logger.
Lyophilization: Freeze-dried PTH (1-34) formulation was used in moisture sorption analysis, TGA, and KF titration as the reference material. Freeze drying was performed using a LyoStar II lyophilizer (FTS Systems, Stone Ridge, NY) using the following cycle: freezing at -40ºC for two hours; primary drying for two hours under the vacuum of 350 mTorr at each temperature of -40,-30,-20,-10, and 0ºC; secondary drying at 10ºC/350 mTorr for 2 hours, 20ºC/350 mTorr for 2 hours, 30ºC/350 mTorr for 1 hours, 30ºC/50 mTorr for 0.5 hours, and 30ºC/0 mTorr for 0.5 hours. Temperature was ramped up at the highest rate offered by the dryer.

Results and Discussion

Challenges of determining water content in ZP-PTH formulation
We previously reported how we overcame various challenges to achieve ≥ 2 year ambient temperature storage shelf life for ZP-PTH based on a liquid formulation consisting of 15.5% w/w PTH(1-34), 16.6% w/w sucrose, 0.2% w/w polysorbate 20, 0.4% w/w HCl, 0.3% w/w NaOH, 0.03% w/w EDTA, 65% w/w WFI at pH 5 [2,3]. It was achieved primarily through the creation of an inert headspace inside the primary packaging (foil pouch) by dry nitrogen purging and including a desiccant sachet (3.5 grams of 3 Å molecular sieves) to absorb the moisture released by the polycarbonate retainer ring (Figure 1b). These procedures created a headspace with low levels of moisture (<1% RH) and oxygen (<1%) [2]. Theoretically, the moisture content of the ZP-PTH final product should equilibrate with the percent relative humidity (%RH) of the storage environment, a relationship called the moisture sorption isotherm (Figure 3) which allows the formulation’s water content to be predicted if the storage humidity is known. Thus, inside the pouch with headspace moisture of <1% RH, the water content of the ZP-PTH formulation should be lower than 1%.
Figure 3: TGA thermograms, % weight lose and derivative weight (°C / %), for lyophilized PTH(1-34) formulation (a) and for PTH(1-34) formulation coated on the titanium array (b), heated at 20°C/min.
Further, the amount of the formulation is small. With 40 mcg PTH(1-34) coated on the microprojection array (the primary dose for Phase 2 and 3 human trials), the total weight of the dry formulation is approximately 80 mcg which is uniformly distributed over the tip of ~1,300 microprojections (i.e., ~0.06 mcg per microprojection) on the titanium array. The array itself has a weight of ~15 mg. Overall, the challenges in determining the water content of the coated array are: the amount of the water may be less than 0.1 mcg; the water content may be <0.01% of the total sample weight (including the titanium array); the formulation volume may be <1% of the total array volume.
Thermogravimetric analysis
TGA is a sensitive water-determining method, capable of detecting down to 1 mcg of weight change, depending on the sensitivity (or detection limit) of the microbalance. During heating water evaporates from the sample normally around 100ºC, which may shift due to the interactions of water with drug/excipients in the formulation as bound or unbound water. However, for a formulation containing other volatile compounds, the applicability and the accuracy of TGA for water determination may be compromised if other volatile compounds interfere with water evaporation.
TGA for PTH (1-34) API: The PTH (1-34) API was manufactured via solid-phase synthesis, multiple preparative HPLC purification, and lyophilization into a PTH(1-34) acetate powder. The API used in this study (Lot #FPTH0501) has a water content of 9.0% and acetate content of 5.1%. The API powder was heated from room temperature to 600ºC at two heating rates, 10ºC/min and 20ºC/min. Two thermograms, % weight loss (from sample’s initial weight as 100%) and derivative weight (% /ºC) as the function of temperature, were plotted. Both heating rates showed four weight-loss phases. The peak for each phase change occurs at 33.6ºC (3.82% weight loss), 119.4ºC (5.99%), 229.0ºC (3.23%), and 314.3ºC (69.9%) at a heating rate of 10ºC/min (Figure 2a) while at 20ºC/min heating rate the thermograms (Figure 2b) exhibited peaks at 36.0ºC (6.24%), 118.3ºC (6.03%), 232.3ºC (3.16%), and 330.1ºC (66.2%). The results are similar but not identical, suggesting that weight loss (evaporation and decomposition) may be affected by the rate of heating. To be consistent, the 20 ºC/min heating rate is consistently used hereafter.
Assigning the volatile species to these weight loss peaks is not straightforward, particularly for water and acetic acid, whose boiling points are close, 100ºC and 118ºC, respectively. The first two weightloss phases might be related to water and acetic acid while the peaks beyond 200ºC might be the result of peptide decomposition. The first weight-loss phase is quite interesting; it occurred at a low temperature, ~30ºC, and weight loss began as early as when heating was just started at room temperature. To identify the volatiles responsible for these weight-loss phases, API samples were heated at 20ºC/min in TGA to 60ºC and 160ºC, respectively. Each sample was then analyzed by RPHPLC for acetate content. After being heated to 60ºC, the API was found to contain 5.2% acetate, comparable to the API before heating, suggesting that it is primarily water (confirmed by KF titration), not acetic acid, lost in the first phase. In contrast, most of the acetic acid was volatized after the sample was heated to 160ºC as its acetate content decreased to 0.3%. With the first three phases accounting for ~15.4% weight loss (Figure 2b) and acetic acid primarily lost in the second phase, it suggests that water evaporated over a wide temperature due probably to different degrees of interaction (binding) force of water molecules with the peptide in the API. Without a clear-cut weightloss phase, it makes accurate water determination by TGA difficult.
TGA for lyophilized PTH (1-34) formulation: The API, formulated with excipients such as sucrose, EDTA, and polysorbate 20 (see composition above), was freeze dried. The lyophilized powder (6.05 mg), with a water content of 5% as determined by KF titration, was subjected to TGA analysis and heated at the rate of 20ºC/min. The thermograms (Figure 3a) are similar to those for API with four weight-loss phases. The third phase, peaked at 212.9ºC, is much more prominent and might be associated with sucrose decomposition. As expected, the first two phases are less visible relative to those measured from the API, which makes reliable water determination even more difficult.
TGA for PTH (1-34) formulation coated on titanium array: The titanium array coated with 80 mcg PTH (1-34) formulation was folded to fit into the sample pan for TGA analysis. The total sample weight is 14.65 mg, mostly contributed by the weight of titanium array itself. The thermograms (Figure 3b) showed only two weight-loss phases, peaked at 210 and 330ºC. The first two weight-loss phases are not visible at all, even after magnification, because the amount of water and acetic acid is very small compared to the total sample weight. This drawback could not be corrected by combining multiple array samples. Thus, it is impossible for TGA to detect the water content of the ZP-PTH formulation directly from the coated system.
Karl fischer titration
KF Titration for lyophilized PTH(1-34) formulation: The freeze-dried PTH(1-34) formulation samples were allowed to equilibrate in the desiccators of 10%, 30% 47% and 60% RH overnight and the amount of moisture in the powder samples was measured by Karl Fischer analysis to be 1.5%, 4.2%, 7.3%, and 11%, respectively. These data perfectly match the moisture sorption isotherm curve in Figure 4, suggesting that the water content can be predicted by the moisture vapor isotherm provided that the storage humidity of the sample is known. Thus, equilibrated in the headspace of <1% RH, the dry formulation, including ZP-PTH coated arrays, should have a water content of <1%.
Figure 4: Overlay of moisture adsorption and desorption data (solid and dashed line, respectively) and KF moisture data generated from freeze-dried PTH(1-34) formulation powder ( ♦ ) and PTH(1-34) formulation coated arrays (⚫).
Water content of PTH(1-34) formulation coated on microprojection arrays: Drying the ZP-PTH formulation is a twostage process: 1) primary drying taking place during/after coating prior to packaging and 2) secondary drying occurring inside the sealed package (foil pouch) in the presence of desiccant. During coating, the microprojection arrays are dipped into the thin film on the rotating drum sitting on the liquid formulation-containing reservoir, and the amount of coating is controlled by the number of dips (passes) through the drug film [4]. The reservoir is controlled at 1ºC to maintain the liquid formulation’s physical stability (i.e., preventing gelation). Since the reservoir is open to the ambient environment, it is important to prevent moisture condensation into the reservoir, which would dilute the formulation and compromise coating uniformity. Thus, the environment surrounding the reservoir needs to be controlled at the dew point (i.e., a temperature which a given parcel of air must be cooled, at a constant barometric pressure, for water vapor to condense into water) to the reservoir at 1ºC. The relationship between air temperature, air relative humidity, and the dew point can be theoretically calculated using a dew point calculator,such as those available online [16]. For an air temperature of 20ºC, to reach the dew point of 1ºC, the ambient air’s relative humidity needs to be controlled at ~30%.
Between each dip, the coated liquid was dehydrated via the ambient air of 20ºC and ~30% RH. The coated liquid is estimated to be dried within a few seconds because of the large specific surface area of the coated formulation, i.e., surface area per unit volume. The theoretical water content of the coated formulation should be ~4% according to the moisture sorption isotherm (Figure 4). However, the actual water content may be higher than 4% because there is probably not enough time for the coated array to reach equilibrium with the ambient air before being packaged inside the pouch. Thus, desiccant (3Å molecular sieves) was included in the final package as a secondary drying measure to not only complete the overall drying process but also maintain the low-humidity headspace inside the pouch for longterm storage. Internal vapor analysis was performed and confirmed that the headspace contains a very low %RH, <1% [20]. Given this information, it is anticipated that the amount of water in ZP-PTH formulation may be <1 mcg (i.e., <1% of the 80 mcg formulation weight).
KF Titration for PTH (1-34) formulation coated on microprojection arrays: According to the operating manual, the detection limit of the KF assay is 10 mcg of water and the sample should contain at least 50 mcg of water for reliable measurement [21]. Therefore, it is required that the arrays are coated with higher doses or multiple arrays be combined to generate samples with enough water for reliable KF measurement. Two high PTH (1-34) doses, 538.1 mcg (1076.2 mcg formulation weight), and 717.7 mcg (1435.4 mcg), were coated on different designs of microprojection arrays. Each coated array was placed in the vial under the ambient condition and allowed to equilibrate in a dew-point controlled hood (at ~30% RH) to simulate the “pre-packaging” condition. The “post-packaged” system represents a coated array packaged with desiccant inside the nitrogenpurged, heat-sealed foil pouch. Sample preparation, such as opening the pouch, transferring the array into a glass vial, and crimping and sealing the vial, was performed inside a glove box controlled at <10% RH by purging with dry nitrogen. To exclude water contributed from non-formulation sources (e.g., array, adhesive, vial, etc), a control sample (uncoated array sample) was prepared and measured with each sample measurement. The results are presented in Table 1.
Table 1: Dietary content,physical activity, weight and blood parameters in 2x1 week diet intervention with low (LFD) and high fibre (HFD) diets in seven healthy men1.
The average water content for the “pre-packaging” samples was ~5% water while that for the “post-packaging” condition is significantly lower, <1%. This result is consistent with the moisture sorption isotherm (Figure 4). However, the water content difference between the sample (coated array) and the control (the uncoated array) is small, <10 mcg, and is close to or actually smaller than the standard deviation of the measurement for both sample and control,suggesting that most of the measured water came from sources other than the formulation. Therefore, KF measurement in this case is very sensitive to the environment, which may easily compromise the accuracy and precision of the method despite a careful sample preparation procedure.
A separate measurement was made in a vial containing 12 coated arrays each coated with a dose of interest, 39 mcg peptide (or ~78 mcg formulation). To minimize water absorption during sample preparation, each pouch containing the coated array should be opened inside a humidity controlled glove box. Unfortunately, opening >10 pouches and placing each coated array into a vial for extraction could take more than one hour, which is difficult and impractical. Thus, each pouch was opened under the ambient condition. After 12 coated arrays were placed in a vial, the whole vial was equilibrated in a low humidity environment (≤ 1.4% RH) created using a desiccator containing 3Å molecular sieves to simulate the “post-packaging” condition. Some vials were equilibrated at 60% RH in a humidity controlled chamber. Again, a control sample, i.e., a vial containing 12 uncoated arrays, was prepared and measured by KF against each sample. The results are listed in Table 2 with each water content number reported as an average of n=3 sample/control sets.
Table 2: KF content for arrays coated with 40mcg PTH stored under 1.4% and 60% RH (n=3).
mThe effect of storage humidity on the moisture content of the coated solid is evident by the increased water measured for samples stored at 60% R.H. (~11.4%), where the amount of water measured from both controls and coated arrays is substantially higher than the 50 mcg threshold and their difference is significantly greater than the standard deviation of the measurement for both sample and control; therefore, KF measurement for systems with such a high water content should be accurate and precise. However, the water content for systems stored in the 1.4% RH environment (i.e., “post-packaging”) is very low (<1%) and is on the same order of the controls (below the standard deviation for both sample and control), confirming the finding with the previous measurements. The water content from these two %RH conditions is again consistent with the moisture sorption isotherm (Figure 4).
It is obvious that measuring the low water content of ZP-PTH by KF titration is a daunting task in terms of pooling a large number of coated systems for each measurement, requiring control samples, and a labor-intensive sample preparation procedure. It is anticipated that >20 coated arrays (and uncoated arrays) are required for a single measurement. Handling these many arrays inside the dry box (opening the pouch and placing each array in a vial) is very difficult. More important, sample preparation is performed in a dry environment to simulate the %RH inside the sealed pouch. The samples eventually equilibrate with the new environment and the measured water content does not necessarily reflect the true storage condition inside the pouch. Therefore, despite all the effort, the value of directly measuring the water content of ZP-PTH is not high. Since the water content can be effectively predicted by the moisture sorption isotherm, the strategy should be shifted to find an alternative method to direct water determination.
Alternatives to direct water determination
To ensure a low moisture level, say, <5% RH (a tentative specification to be confirmed with stability data), inside the pouch, desiccant capacity (or performance) need to be verified. Pouch seal integrity reflects the effectiveness of the nitrogen-purging and heatsealing process and can be confirmed by a low level of oxygen content (<1%) in the headspace. Determination of desiccant capacity is to ensure the desiccant can pull the headspace moisture level inside the pouch to below 5% RH. Two approaches were proposed and discussed below. Please note that pouch seal integrity should be validated and is an important part of packaging performance tests, which is beyond the scope of this investigation.
Internal vapour analysis (headspace analysis): This method directly measures moisture and other gases in the headspace inside the pouch. The pouch sample is placed in a test chamber sealed against a VitonTM O-ring. A pin pierced the pouch through the center of the O-ring to take a headspace sample, which was then subjected to a mass spectrometer for water vapor analysis. This method was used to assess the performance of 3Å molecular sieve desiccant in two formats, a 3.5 gram sachet or a 1.0 gram desiccant co-molded into the retainer ring (Figure 1b). Despite the fact that the co-molded desiccant ring has a lower capacity, both forms of desiccant performed effectively, reducing the moisture level in the headspace to <100 ppm (<1% RH). This headspace method requires expensive apparatus and method validation for official QC testing. These two desiccants performed differently in the rate of dehydration; it took less than one hour for the 100% capacity (fresh) desiccant sachet, vs. approximately 20 hours for the 100% capacity co-molded desiccant ring, to reach below 1% RH (Figure 5). The moisture reduction profiles were measured by a Datatrace II moisture probe sealed inside the pouch. Although using such a probe is easy and straightforward, it’s impractical as the automated packaging process needs to be interrupted for probe insertion.
Figure 5: The profiles of headspace moisture (%RH) reduction vs. time measured by a data tracer (moisture monitor sealed in the pouch) for four types of desiccant: 100% capacity 3.5-g desiccant sachet, 100% capacity co-molded desiccant ring, 65% capacity co-molded desiccant ring, and 50% capacity co-molded desiccant ring.
Desiccant capacity determined by weighing: Although it is confirmed that the incoming desiccant with 100% capacity can effectively remove the headspace moisture, desiccant capacity may deteriorate upon exposure to the manufacturing environment. The manufacturing facility is controlled at ≤ 25ºC and ≤ 60% RH and the exposure time is expected to be less than 24 hours. Therefore, desiccant capacity needs to be confirmed after such exposure. With a fresh sample capable of adsorbing 100 mg water, the capacity of the co-molded desiccant ring can be easily quantified by the weighing method. By exposing the desiccant co-molded rings to an accelerated condition, i.e., 40ºC/75% RH, and monitoring the weight gain (as the result of moisture uptake), it was found that the desiccant capacity was depleted by 35% (or 65% capacity due to 35 mg weight gain) in 16 hours and by 50% (or 50% capacity due to 50 mg weight gain) in 24 hours, which represents the worst-case conditions as the same desiccant co-molded ring was depleted by only 10% upon exposure to the controlled manufacturing environment, 25ºC/60% RH, for 24 hours. Each of the 35% and 50% depleted rings was pouched with a Datatrace II moisture probe to monitor the headspace moisture reduction profile inside the pouch. The profiles in Figure 5 suggest that the 50% and 65% capacity rings adsorbed moisture at slower rates than the 100% capacity co-molded rings. After 400 hours, the headspace moisture reached 3% and 4% RH for the 65% and 50% capacity rings, respectively. The 50% capacity ring still satisfies the 5% RH threshold and is considered acceptable. Thus, a QC method can be developed to test the incoming co-molded desiccant rings by exposing the rings for 24 hours at 25ºC/60% RH. With a weight gain of less than 50 mg, the desiccant rings should have sufficient desiccant capacity to maintain the headspace moisture below 5% RH, thereby ensuring a low level of water in the formulation.


This study applied creative approaches to investigate the feasibility of determining the water content of the coated ZP-PTH formulation using TGA and KF titration. TGA is not a feasible methodology because water loss during heat scan is not a single-phase event and is interfered by another volatile compound, acetic acid. More important, the weight of the titanium array itself exceedingly outweighed the water amount in the formulation. TGA is simply not sensitive enough for the individual coated arrays, and this problem could not be overcome by pooling multiple coated array samples. Although KF titration allowed pooling >20 array samples to overcome its sensitivity challenge, the sample preparation procedure, working in the dry box, the need in preparing control samples, and conditioning the samples to a pre-determined condition during sample preparation, made KF titration impractical as a routine product release method. More important, direct KF measurement did not yield more information than what’s predicted by the moisture sorption isotherm. We proposed two approaches capable of ensuring the formulation’s moisture content without directly measuring the water content. Overall, this study presents an example of adopting an outsidethe- box thinking to characterize a novel transdermal product with a unique dosage form, which is otherwise difficult to satisfy the regulatory requirement.


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