Abstract
The stability of polygeline-based blood plasma expanders Haemaccel and Gelofusine were examined in context of their sensitivity to environmental factors, because drug stability is critical element in accurate and appropriate delivery of drug therapy to patients. This study was initiated to specifically and critically assess stability of Haemaccel and Gelofusine according to ICH guidelines with the aim of delivering safe, appropriate, acceptable, and efficacious administration of drug product in any situation. This study revealed that Haemaccel and Gelofusine are suitable for storage at different temperature and at different storage conditions until its expiry date, shelf life, or utility time, for their quality, safety, suitability, acceptability, and efficacy.
LAY ABSTRACT: Stability studies of two different plasma substitutes, Haemaccel and Gelofusine, were examined according to ICH guidelines for their expiry or utility time, because drug stability is very important element in accurate, suitable, and correct delivery of drug therapy to patients. The aim of present study is to deliver the safe, appropriate, acceptable, and right drug product in any situation. The results indicate negligible changes in different parameters during stability study, except for pH and viscosity. This study shows that both drug products are suitable for storage at different temperature and at different storage conditions till their expiry date or utility time, for their quality, safety, suitability, acceptability, and strength.
Introduction
Proteins or polypeptides and polysaccharides containing drug products such as plasma substitutes are particularly sensitive to environmental factors; therefore, these have become an important class of potent therapeutic agents in plasma substitutes in the last two decades due to their unique physicochemical and biological properties. However, proteins are marginally stable and highly susceptible to both chemical and physical degradation (1⇓–3). Therefore, stability issues are very important in protein and polysaccharides containing plasma substitutes due to prolonged storage. The aim of this study was to determine the stability of Haemaccel and Gelofusine at different temperature and at different storage condition such as at room temperature, at 40 °C, in freezing conditions, and in exposure to direct sunlight.
Materials and Methods
Materials
The studies were carried out at room temperature, at 40 °C, in freezing conditions, and in exposure to direct sunlight. Different batch no. E073, E074, E080, N036 of Haemacel and Gelofusine batch no. 4212E41 were tested for stability studies (Table I). Haemaccel batch no. E073 and E080, Gelofusine batch no. 4212E41 were kept in a Binder climate chamber (model KBF720, Bohemia, NY, USA) for stability studies at room temperature and at 40 °C.
A pH meter WTW 525 (Wissenschaftich-Technische Werkstätten, Germany) was used for measuring pH during stability studies of Haemaccel and Gelofusine. Free amino groups were assessed using a Mettler DL40RC Memo Titrator and a WTW 525 pH meter. The relative viscosity of Haemaccel and Gelofusine was determined at 35 ± 1 °C using an Ostwald viscometer (capillary viscometer, Schott AG, Germany). Total nitrogen was determined by the Kjeldahl method using a Buchi 426 digestion unit and Buchi 339 distillation unit during studies. Electrolytes were detected by a previously calibrated flame photometer PFP7 (Jenway, England) against freshly prepared standard solution. A PCLM3 chloride meter was used to measure chloride. A calibrated pyrometer (Ellab APT75) was used to determine whether Haemaccel/Gelofusine was pyrogen-free. Sterility was determined with previously cleaned and sterilized filter assembly.
Methods
The following physical, chemical, and microbial parameters were evaluated after completation of storage time: appearance, pH, viscosity, free amino groups, nitrogen content, electrolytes, loss in weight, pyrogen, and sterility. All glassware and utensils were cleaned with chromic acid and then rinsed with distilled water. Before starting the stability studies tests, all relevant instruments were cleansed and calibrated for accuracy.
Appearance was assessed visually for signs of precipitation or other evidence of alteration such as turbidity, haziness, or changes in color. A WTW 525 pH meter was used for measuring pH during stability studies of Haemaccel and Gelofusine.
The relative viscosity of Haemaccel and Gelofusine was determined at 35 ± 1 °C using an Ostwald viscometer (capillary viscometer, Schott). The flow rates of 5 mL distilled water and then 5 mL Haemaccel and Gelofusine were determined, respectively.
Free amino groups were assessed using a Mettler DL40RC Memo Titrator and a WTW 525 pH meter. In this test a mixture of 40 mL Haemaccel /Gelofusine and 1.6 mL 2N hydrochloric acid was titrated up to pH 6.00 using 0.2N sodium hydroxide. The quantity of sodium hydroxide consumed (M1) was recorded; 4 mL of formaldehyde solution (Merck) previously adjusted to pH 7.00 ± 0.02 was then added. The pH value fell sharply after 1 min. The titration was continued to pH 8.50 and the consumption of sodium hydroxide (M2) was recorded. Free amino groups were calculated using the formula: (M2 – M1)/5F = mL of 1N sodium hydroxide/40 mL of Haemaccel/Gelofusine, where F is a factor of 0.2N sodium hydroxide.
Total nitrogen was determined by the Kjeldahl method using a Buchi 426 digestion unit and Buchi 339 distillation unit during studies. Haemaccel/Gelofusine (1mL) plus concentrated sulphuric acid (15mL; Merck) and one Kjeldahl tablet, batch no. TP707758 (Merck) were digested for 30 min at 650 °C in the digestion unit. After digestion, the sample was cooled and titrated using the distillation unit. Before titrating the sample, the pH electrode in the distillation unit was calibrated using buffer solution pH 4, batch no. OC354850, and buffer solution pH 7, batch no. OC354838.
Electrolytes were detected by previously calibrated flame photometer PFP7 (Jenway) against freshly prepared standard solution. The preparation of test and standard solution was as follows. The sample/test solution was 1 mLHaemaccel/Gelofusine in 100 mL distilled water. To prepare the standard solution, 1000ppm 3.34 mL Na +, batch no. FINA4HI (Jenway), 1000ppm 0.20 mL K+, batch no. FIK2LI (Jenway) and 1000 ppm 0.25 mLCa++, batch no. FKA12MI (Jenway) were mixed in a 100 mL volumetric flask and remaining volume filled with distilled water. A PCLM3 chloride meter was used to measure chloride. Calibration and determination of chloride was as follows: 5 mL acid buffer solution, batch no 1273 (Jenway), 10 mL distilled water and 0.3 mL or 10 drops of gelatin solution, batch no 2237 (Jenway) were placed in plastic beaker with a stirrer bar and the beaker was placed on the instrument platform. The 20 μL range was selected and 20 μL of a standard solution of chloride 100mmol/L, batch no. 0250135CI (Jenway) was added and conditioning was performed. After conditioning, a further 20 μL of a standard solution was added and then titrated; the reading was close to 100mmol/L. After calibration the whole process was repeated in the same way, except 20 μL Haemaccel was added instead of standard solution and the reading were recorded.
Loss in weight was determined by recorded initial weight (W1) of Haemaccel and Gelofusine bottle when the study was started and the different storage time weight (W2) during the study—1, 2, 3, 6, 12, 24, and 36 months—at different temperatures: room temperature, at 40 °C, and in a freezer (−5 to −15 °C) by using the formula
A calibrated pyrometer (Ellab APT75) was used to test whether Haemaccel/Gelofusine was pyrogen-free. In the pyrogen test, three pretested rabbits each received 10 mL Haemaccel/Gelofusine per kilogram body weight intravenously. Temperature rises were interpreted according to the British Pharmacopeia IV (BP).
Sterility was determined by a previously cleaned and sterilized filter assembly according to the European Pharmacopeia (Ph.Eur.) (2.6.1). About 200 mL of Haemaccl/Gelofusine was filtered through the filtration assembly by using a suction pump. After filtration the membrane filter (Cellulose nitrate, diameter: 50 mm, pore size: 0.45 micron, Ireland) was cut into two equal halves and one half transferred into soya broth medium and the other into thioglycollate medium. Observation time of incubation for both soya bean casein digest medium (at 20–25 °C) and for fluid thioglycollate medium (at 30–35 °C) is 14 days.
Results and Discussion
Stability Results
Stability study of Haemaccel and Gelofusine at various storage conditions showed considerable change in pH value and in viscosity. Different scientists also reported change in pH value and in viscosity, such as Gabr (1996) and coworkers, who reported decreased value in pH (7.3 to 7.0) and in relative viscosity (2.18 to 1.98) of oxypoly gelatin (4). Theiercelin also reported that storage temperature affected the viscosity of gelatin, polyvinyl pyrrolidone, and dextran (28). The appearance of Haemaccl/Gelofusine remained the same during the stability study. Stability of Haemaccel at room temperature for 3 years showed decreased values of viscosity and pH from 1.76 and 7.13 to 1.68 and 6.89, respectively. Increased value of free amino groups (from 0.525 to 0.591) and loss in weight (from 0.10% to 0.83%) and fluctuation value was observed in nitrogen contents. Loss in weight gradually increased in this study. The results of pyrogen and sterility were satisfactory (Table II). During stability study at 40 °C for 6 months, decreased value of viscosity (from 1.77 to 1.65) and pH (from7.28 to 7.13) were recorded. The value of viscosity sharply decreased, which was considerable.
Increased value of free amino groups (from 0.527 to 0.578)—which is also reported by Thiercelin et al. (31)—and loss in weight (from 0.21% to 0.35%) and a little decreased value of nitrogen contents (from 6.40 to 6.35) were observed. Results of pyrogen and sterility were satisfactory (Table III). On the other hand, stability study of Haemaccel in sunlight (days and night) for 3 months showed gradually decreased pH (from 7.16 to 6.17) and viscosity (from 1.76 to 1.70). Fluctuations were observed in nitrogen and chloride values. Results of free amino groups were from 0.542 to 0.514, and loss in weight was from 0% to 0.087%. Results of Na+, K+ and Ca++ ions remained almost the same, and results of pyrogen and sterility were satisfactory (Table IV). The stability of Haemaccel in freezing conditions showed a slight change in viscosity (from 1.77 to 1.76), a decreased value in pH (from 7.27 to 7.10), a little change in free amino groups (from 0.544 to 0.562), and loss in weight (from 0.01% to 0.03%); fluctuation was observed in nitrogen but on the whole insignificant changes were observed in this study (Table V). Stability study of Gelofusine at room temperature over a 3 year period showed slightly dropped values in pH (from 7.28 to 7.08) and viscosity (from 2.10 to 1.81), increased values in free amino groups (from 0.555 to 0.641) and loss in weight (from 0.02% to 1.07%), and decreased values observed in nitrogen (from 7.39 to 7.29). Rate of changes in different parameters are less in Gelofusine as compared to Haemaccel, which may be depend on concentration differences (Gelofusine is 6% and Haemaccel is 3.5%) and also the manufacturing process (succinic acid anhydride is used in Gelufusine, whereas hexamethylene di isocyanate is used as a cross-linking agent in Haemaccel). On the other hand, the stability of Gelofusine at 40 °C showed decreased values in pH (from 7.28 to 7.08) and in viscosity (from 2.10 to 1.632), increased values in free amino groups (0.555 to 0.739) and loss in weight (from 0.109% to 1.23%), and decreased values observed in nitrogen (from 7.39 to 7.29) (Tables VI and VII). Stability study of both plasma substitutes at 40 °C show that Haemaccel is more stable in the parameters of free amino groups and in loss in weight, as degradation rate is low as compared to Gelofusine.
Discussion
This study was carried out to determine whether different temperature and different storage condition such as at room temperature, at 40 °C, in freezing conditions, and in exposure to direct sunlight would alter the physical, chemical, and microbial parameters of Haemaccl/Gelofusine during stability study. For this purpose various different parameters (pH, relative viscosity, free amino groups, nitrogen content, electrolytes, pyrogen, and sterility) were considered. The results demonstrated negligible changes during stability study except for pH and viscosity, which were considerable. Gabr (1996) and coworker also reported decreased value in pH (7.3 to 7.0) and in relative viscosity (2.18 to 1.98) of oxypoly gelatin. In the same way degraded gelatin solution also decreased its relative viscosity (1.79 to 1.58), but pH value slightly increased (5.2 to 5.4) (4).
Different scientist studied the stability of different drug products in different infusion solutions at different storage conditions, such as Jean-Daniel (2005) and coworkers investigated the effect of freezing, long-term storage, and microwave thawing on the stability of ketorolac tromethamine in dextrose 5% infusion. They observed no color change or precipitation, but pH value decreased slightly (5). Fischer (1997) and coworkers determined stability of fosphenytoin sodium admixtures with NaCl 0.9% injection and dextrose 5% injection storage at room temperature for 30 days. They did not observe any visible precipitation or change in color or clarity during stability study (6).
Mendenhall (1984) has reviewed stability aspects of parenteral products and has shown that discoloration often is either photochemical or oxidative; sometimes a cloud or precipitate may appear in drug products as storage time progresses. This is most often due to chemical changes in the system (7). There was not any change observed in appearance of Haemaccel and Gelofusine in this study during different storage conditions. Pale yellowish color was observed in Haemaccel during stability study in sunlight. But there is not any change of color observed in Haemaccel and Gelofusine during different storage conditions: at room temperature, at 40 °C, and in freezing conditions (−5 to −15 °C). Neuwald reported water loss in Haemaccel during storage up to 3 years at temperatures between −20 and +60 °C (8). In our study slightly decreased values were observed in loss in weight in both Haemaccel and Gelofusine at room temperature and at 40 °C. Viscosity of liquid usually decreases with rise in temperature. The amount of such a decrease is often of order of 1% to 10% per degree Celsius.
The opposite effect may occur in certain cases, such as aqueous solutions of synthetic polymers like methylcellulose, which exhibit gel formation when temperature is increased (9). Theiercelin et al. (28) examined the viscosity of plasma substitute solutions and found that storage temperature affected the viscosity of gelatin, polyvinyl pyrrolidone, and dextran solutions. In further study Thiercelin et al. (29) observed the effect of supersonic waves on viscosity of plasma substitute solutions. They concluded that ultrasonic waves have little or no influence on viscosity of solutions. The present study also conforms the stated results of Theiercelin that storage temperature affects the viscosity, as Haemaccel and Gelofusine also showed decreased value of viscosity, at room temperature and at 40 °C on duration of 3 years, 6 months, and 30 months (in freezing condition) in Haemaccel, and 3 years at room temperature, 30 months at 40 °C in Gelofusine in this study respectively; but at storage in sunlight quite little change of results was observed during study of Haemaccel under sunlight for 30 days. But it was also observed that two different type of acetyl starch (AS299 and AS297) showed a constant relative viscosity over a period of time of 140 days (10). Siragusa in 1955(11) demonstrated that increased viscosity makes an emulsion more stable. Hetastarch has a viscosity of 4.5 kg.m−1·s−1 and Haemaccel 1.23 kg·m−1·s−1or Pa·s. The present study also confirmed the study of Siragusa, as viscosity of Haemaccel was decreased. But on the other hand, stability study of Haemaccel in freezing conditions (−5 to −15 °C) showed slightly decreased value of viscosity during 3 years, which conformed to the claim of the manufacturer.
The most important factors that influence the rate of drug decomposition in drug delivery systems are solution pH and temperature. The stability of many disperse systems, and especially of certain emulsions, is often pH-dependent. Therefore, drug reaction rates are generally less at intermediate pH values than at high or low ranges, and most drugs are sufficiently stable in pH range of 4 to 8. For example, morphine solutions do not decompose during 60 min exposure at a temperature of 100 °C if the pH is less than 5.50 (12). During the Cuban crisis, the American supplies of liquid dextran solutions were examined and scientists found change in pH values, diminished vacuum, and a flaky white precipitate for up to 10 years in storage conditions (30). But Cadrobbi coworker reported that no color change or precipitation occurred in sodium folinate in 5% dextrose for a period of at least 30 days at 48 °C, but the pH values of infusion solution decreased slightly without affecting chromatographic parameters (13). The present study also showed change in pH values of Haemaccel and Gelofusine during storage of 3 years at room temperature and at 40 °C. Haemaccel storage in freezing conditions (−5 to −15 °C) showed a little change in pH value. Lebitasy et al. also reported slight change in pH values (6.52 ± 0.01 to 6.50 ± 0.01) of calcium levofolinate in 5% dextrose solution stored at 5 ± 3 °C for 1 month (14).
On the other hand, Haemaccel stored in sunlight for 30 days showed a gradual decrease in pH values, which is supported by the above study. Carboxylic acid ester, amides, and imines are labile to hydrolysis. As polygeline is manufactured by bovine gelatin, it consists of different polypeptide bonds, bound together to form urea bridges by cross-linking with hexamethylene di-isocyanate. It has different side groups, which are reactive groups such as hydroxyl groups, amino groups, carbonyl groups/carboxyl; these groups react with isocyanate group of hexamethylene di-isocyanate.
Amino groups react to form urea derivatives, where as they react with hydroxyl groups to form carbamic acid esters. Free carboxyl groups react with free amino groups to form peptide bonds, and esters groups react in known manner with lysine- α amino groups, forming cross-linking peptide bonds. Or, if amine and carboxylic acid functional groups in amino acids join together to form amide bonds, a chain of amino acid units is formed called peptide, and connected by C-N bonds (covalent) will produce water.
As esters are rapidly degraded in aqueous solution and polygeline (Haemaccel) also contain water for injection (WFI) or (make up with water for injection), so it is possible that hydrolysis causes breaking of polypeptide chains because the presence of hydroxyl groups produce free amino groups. In the same ways extreme temperature/heat will result in the unfolding of polypeptide chains, leading to change in structure and often a loss of function. Covalent interactions between amino acid side chains of ploypeptide are lost, there is breaking of C-N, and free amino groups are formed. At low-pH levels, protein will denature due to loss or gain of protons, and will lose their charge or become charged. This will eliminate ionic interactions and may cause breaking of linkages of polypeptide bonds.
Thiercelin et al. reported that high temperature during storage of plasmagel caused a slight increase in fluidity and acidity. He recommended that it should be checked periodically (31). The present study also showed increased value of free amino groups in both Haemaccel and Gelofusine during storage at room temperature and at 40 °C (Table II and III). The amphiphilic nature of the protein molecules results in their adsorption to a wide variety of surfaces and also in their loss and destabilization (15⇓⇓⇓⇓–20). Side chains of tyrosine, phenylalanine, and tryptophan, as well as peptide bonds in proteins, absorb ultraviolet light. Both ionizing and nonionizing radiation can cause protein inactivation (21).
Haemaccel stored in sunlight for 3 months as a protein formulation has poor photostability because many amino acid residues are prone to photolytic degradation (22). Our study also showed increased value of free amino groups because heat is probably the most common cause of disruption or unfolding of protein's natural secondary or tertiary structure, leading to denaturation (23). In the same way, a little change was observed in Haemaccel's free amino groups during storage in freezing conditions up to 3 years.
Neuwald reported the results of Haemaccel storage up to 3 years at temperature between−20 and +60 °C (8). His findings showed variations in nitrogen values during storage. The present research also supports the Neuwald (8) study, that is, fluctuation was observed in nitrogen content during storage of Haemaccel at room temperature, at 40 °C, in sunlight, and in freezing conditions. Gelofusine showed the same results as Haemaccel, that is, fluctuation. Several important drug interactions occur as a result of therapeutic agents altering concentrations of electrolytes, such as potassium and sodium. When these drugs are included in therapeutic regimen, it is important that electrolyte levels be periodically monitored. Electrolytes also play important role in the stability system. If there is a greater concentration of electrolytes then there is a greater stability of the system.
In this study, results of electrolytes values were almost similar during stability study. The problem of pyrogenic reactions to intravenous injection had been observed as early as in 1911 when Wechselman noted an increase in temperature and chills in patients receiving injections of arsphenamine. In 1923, Florence Seibert discovered that the drug fevers referred to by Wechselman were caused by bacteria-produced pyrogens. Her studies were extended to the development of the rabbit test for pyrogens, still the USP test method. While looking for a quicker and simpler pyrogen test for radiopharmaceuticals, Cooper and his associates, in 1969, developed the limulus test, a test for bacterial endotoxin using Limulus amebocyte lysate (24). The rabbit or pyrogen test, along with a sterility test, become the two most important tools of the pharmaceutical industry. The pyrogen test employing rabbits is still in limited use; an endotoxin test using an extract from blood cells of horseshoe crab is the predominant pyrogen test today.
In the present study the pyrogen test was employed. Pyrogen testing is incorporated as a released criterion for the product, but unlike sterility, it is rarely used as a test criterion after release of product. During storage at room temperature, at 40 °C for 6 months, and in freezing conditions for 30 months, Haemaccel and Gelofusine remained pyrogen-free.
For certain LVP solutions in plastics, a sterility check is incorporated into the stability protocol to verify the integrity of container at specific intervals. The sterility test or procedure will detect susceptible areas of the container, potential problem sites—for example, loosening of latex plugs at medication site, air-inlet site, or improperly sealed rubber closures—and caps of bottles, as well as improper molding of bottles. This study revealed that during storage of 3 years at room temperature (Haemaccel and Gelofussine), 6 months and 30 months at 40 °C (Haemaccel and Gelofusine respectively), and 3 years storage in freezing conditions (Haemaccel), both plasma substitutes are maintained their sterility.
Lundsgaard-Hansen and coworker reported that solutions of 5–6% dextrose, 0.9% saline, and Ringer's lactate are stable for up to 1 year or more, even if stored at ambient temperatures. Dextrose solutions may acquire a yellowish tinge (caramelization), but this is clinically insignificant.
The dextrans and the gelatins are very sensitive to temperatures exceeding 20–30 °C. A degradation into smaller molecules begins after 1 month of storage at 40–60 °C and is very marked (approximately 30–60% decrease in the average RMM) after 5–6 months. In clinical terms, this would shorten their intravascular volume effect and accelerate their renal elimination.
In contrast, hydroxyethyl starch 450 shows no such degradation after 6 months of storage at 40–60 °C. Although heat and pH are the main factors that can cause aggregation (25), they can lead to precipitation of protein (26). But prolonged storage under adverse conditions may result in a crystalline precipitate or a deep brown, turbid appearance in starch solution (12). Long periods of storage or exposure to temperature fluctuations may cause the formation of flakes in dextran solution (4). Acetyl starch is not stable in long-term storage because acetic acid is separated in large amounts at 20 °C and 40 °C (10). In hot climates, colloidal plasma substitutes should not be stored at ambient temperatures for periods exceeding 1 month, and they preferably should be stored at temperatures below 20 °C. If stored at refrigerator temperatures, they should be warmed prior to use to avoid hypothermia in the recipient (27).
All of these observations and results are useful for storage of these plasma substitutes at different temperature for their expiration time, shelf life, or utility time restriction for hot and humid region of world.
Conclusions
This study revealed that Haemaccel and Gelofusine are suitable for storage at different temperatures and at different storage conditions until their expiry date, shelf life, or utility time, for their quality, safety, suitability, acceptability, and efficacy.
- © PDA, Inc. 2013
References
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