Comparison of pharmaceutical quality of eight generic ganciclovir injections in China and Cymevene
ABSTRACT
To assess the pharmaceutical quality of eight commercially available generic products of ganciclovir injection produced in China with original brand product (Cymevene, Roche, Switzerland). Tests were performed according to China Pharmacopoeia 2015 and Import Drug Registration Standard introduced by CFDA. Items including characteristics of the packing and reconstituted solution, pH, visible particles, content of active and related substances, sterility and bacterial endotoxin were all carried out based on the standard laboratory operating rules and requirements. Seven of all tested domestically produced generics of ganciclovir for injec- tion failed to reach the in vitro quality requirements in comparison with the original brand product Cymevene. Three generics failed to meet the standards for pH of an aqueous solution. One out of eight generic products fell outside the specifications for API content. All generics showed impurities, whose levels were generally greater than observed in the brand product. One generic product was identified an endotoxin contamination. In addition, six generic products failed to reach the quality requirements of water content, which should be under 3%. Most tested ganciclovir products failed to meet the pharmaceutical quality standards for original brand product. Important items like pH, endotoxin contamination, content of API and impurities could cause clinical attention, as they directly affect the therapeutic efficacy and patient tolerance.
Introduction
Ganciclovir, an acyclic nucleoside analogue of 20- deoxyguanosine, is widely used as an antiviralagent to prophylaxis and cure cytomegalovirus (CMV) infections in immunocompromised patients. Once phosphorylated to ganciclovir triphosphate with the action of viral kinases, viral DNA poly- merases will be competitively inhibited by ganciclo- vir, which can cause the DNA chain terminationand block the replication of herpes simplex virus and CMV.1–4Ganciclovir is regarded as a class-III drug in the Biopharmaceutics Classification System (BCS). Compared to its good water solubility, the perme- ability of ganciclovir across the membrane is poor. Due to its nature of high hydrophilicity and poor permeability, the oral bioavailability of ganciclovir is lower than 7%.5,6 Thus, intravenous infusion is its most appropriate and commonly used administration route. For intravenous administra- tion, ganciclovir is formulated as a monosodium salt form in a white to off-white sterile lyophilized powder. CymeveneVR is the brand name drug of ganciclovir for injection, which is patented and manufactured by F. Hoffmann-La Roche (Roche). It has been used worldwide in the treatment of CMV infections in immunocompromised patients, including AIDS and organ transplant patients.Generic products of Cymevene in many coun-tries are available since the expiration of its patent during 2002–2005. In China, more than 80 generic products of ganciclovir for injection from differentmanufacturers could be found in the market. However, little information has been concerned about the pharmaceutical quality of generic prod- ucts of ganciclovir for injection in China. Generally, for oral formulations, the quality con- sistency between innovator and generic product could be guaranteed through bioequivalence test.7 Unlike that, the active pharmaceutical ingredient (API) in the parenteral formulations will be directly enter blood circulation system, which called complete bioavailability.
Thus, a proof of pharma- ceutical equivalence is assigned for therapeutic equivalence evaluation of parenteral formulations.9 Pharmaceutical equivalence requires that the gen- eric products have the same API, administration route, dosage form and concentration. In addition, generic products should also meet the requirements for brand product like quality, strength and purity described in the compendial and other stand- ards.10,11 Therefore, through affirming pharmaceut- ical equivalence, the generic products could be considered to become a substitute for innovator ones. Actually, pharmaceutical equivalence between some generic formulations and innovator branded products has already been compared, which showed that generics could not reach either inter- national standards or specifications of originaldrugs.9,12–15 These failed tests involved in sterile,visible particles, pH, content of API, related sub- stances and so on. Under these circumstances, the risk of reducing efficacy of API may resulting in clinical failure.16 Meanwhile, thousands of patients are receiving a treatment of generics even with potentially life-threatening risks. Faced with this, ensuring the quality consistency between generic and original products of ganciclovir for injection is of paramount importance before clinical application.In present study, the pharmaceutical quality of Cymevene with 8 generic products commercially available and popular on the market in China was compared, according to detailed quality standard stated in China Pharmacopoeia 2015 and Import Drug Registration Standard introduced by CFDA.purchased from Fisher Scientific Co. (Santa Clara, CA, USA). Ultrapure water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA).Test productsNames, specifications, sources, batch numbers, manufacture and expiration dates of Cymevene and eight generic products are listed in Table 1.Container closure integrityThe vials were firstly immersed in a methylene blue solution, and then reconstituted with water (50 mg/ ml ganciclovir). By visually observation of a blue coloration in the reconstituted solution, the integ- rity of container closure was assessed.Colour of content and reconstituted solution Colour of the content was assessed by visually observation as the ways showed in the ‘MunsellBook of Colour.’
The reconstituted solutions (50-mg/mL ganciclovir) were firstly shifted to 15-mL colourless test tubes, the colour of which was then compared with colour scale described in the European Pharmacopoeia. Colour of content should be white to off-white powder, while the reconstituted solution should be less coloured than Yellow 5.colorColorColorcolorColorcolorcolorFill mass of each vialAfter removing the packaging, five containers were weighted one by one. Then, empty containers were rinsed with ethanol and water, dried at 110 ◦C for2 h, and weighed again until cool to room tempera-ture. The average of weight difference was fill mass. The difference for fill mass between each vial should not exceed ±7%. pH value filtered through membrane. Then the membrane fil-As the method showed in the China ters were incubated in 100 ml trypticase soy broth Pharmacopoeia, the pH value of reconstituted solu- tion was assessed using a pH-meter (FE28-Meter, Mettler Toledo, Switzerland).Visible particles analysisVisible particles analysis was performed by lamp inspection method described in China Pharmacopoeia. 3 vials were dissolved in water (50 mg/ml ganciclovir) and shaken until powder dis- solved completely. After standing for 3 min, visible particles were observed in an appropriate distance (around 25 cm) with gently rotation. Requirement for the test was no more than 3 particles per container.Identification and determination of the content of active and related substancesThe API and impurities contents were identified and determined as the method described in Import Drug Registration Standard by using high-perform- ance liquid chromatography (HPLC).
Separations were accomplished on an Eclipse plus C18 reversedphase LC column (50 × 2.1 mm, i.d. 1.8 lm,Agilent, USA). The mobile phase was methanol and water (methanol:water ¼ 5:95). The sample- tray temperature was set at 4 ◦C, while the flow rate was 1 mL/min and the column temperaturewas kept at 30 ◦C. The retention time of the main peak of the test solution should be consistent withthat of the reference solution. The content of active substances should not exceed its labelled amount of 90.0%-110.0%. In addition, the main impurity guanine should not exceed 1% of the labelled amount. The sum of the peak areas of the impur- ities in the chromatogram of the test solution should not be higher than 1% of the main peak area.Membrane filtration method was used to test steril- ity of all products. The vials were firstly reconsti- tuted with sterile water (50 mg/ml ganciclovir) and and 100 ml trypticase soy broth, respectively. After 14 days incubation, microbial growth was checked. Ten vials of each product were tested.The bacterial endotoxin was determined by gel-clot method described in China Pharmacopoeia. The vials were firstly reconstituted with water for bac- terial endotoxin test (50 mg/ml ganciclovir). Then, the solution of samples and test water were incu- bated with limulus amebocyte lysate, respectively. Negative control without limulus amebocyte lysatewas also tested. The samples were checked after 60 min of incubation at 37 ◦C. The amount of endotoxin should be less than 0.1 EU in 1 mg ofganciclovir. Three vials of each product were tested.
Results
Characteristics of the vials and contentsAs shown in Table 2, similar to Cymevene, the container of each generic product was tightly closed with good integrity, and the colour of vial content was white or off-white. The amount of fill- ing content of all eight generic products fell within the standard (±7%).Characteristics of the reconstituted solutions As shown in Table 3, three generics fell out the standard for pH of an aqueous solution (10.8 ~ 11.4). Two generics were not colourlesscompared as required. Both dissolution time andvisible particles examines of all the products are qualified.API content, moisture and impuritiesAs shown in Table 4, the average API content of one generic fell out the Import Drug Registration Standard (90%–110%). For the moisture tests, sixgenerics failed to meet the standard that the watercontent should be lower than 3%. The total content of impurities of all generics fell within the standard that under 1%. Furthermore, specified impurity of these products, which was identifiable as guanine, did not exceed the standard either. It is worth not- ing that the amount of guanine in the Cymevene was too low to be analysed by HPLC.Sterility and bacterial endotoxinAll generics were sterile. Bacterial endotoxins in all generics were under China pharmacopoeia specifi- cations except one, which exceed 0.1EU/mg.
Discussion
In order to relieve the pressure of increasing healthcare costs, generic products is widely spread and regarded as substitution of brand products by healthcare providers. In theory, using a formula- tion of generic product should avoid any risks to the health of the patient like brand one. However, it is hard to ensure all generic products perfectly achieving the standards on preparation and stor- age, safety and clinical efficacy set by brand prod- ucts. Under these circumstances, agencies have established series of strict guidelines for pharma- ceuticals to guarantee the quality of generics. For the drug products for injections, the pharmaceut- ical quality and consistency among products is set for monitoring the quality of these copies of brand products.The present study was undertaken to provide information about the pharmaceutical characteris- tics of 8 generic ganciclovir formulations. Test including container integrity, colour of vial content, filling content, colour of reconstituted solution, dis- solution time and visible particles of eight generics all fell in the specifications for ganciclovir injec- tions. Unfortunately, seven generics failed to meet the ganciclovir specifications in one or more tests like pH measurement, content of API and impur- ities, endotoxin contamination and residual mois- ture, which would have potential bad impact on the safety and efficacy of products.Three generics fell out the standard for pH of an aqueous solution. The Roche specifications for ganciclovir injections have emphasized that the solution of Cymevene must be infused into veins with adequate blood flow with an infusion time over 1 h. This is due to the high pH of ganciclovir solutions may result in severe tissue irritation and cause phlebitis and/or pain at the site of IV infu- sion.17 Therefore, the pH of ganciclovir solution represents a true quality issue and should be strictly controlled.The real API contents in two generic products had deviated from their labeled amount (250 mg) more than 15 mg, and one of them even fell outside the specifications for API content, while API con- tents in Cymevene fit much well (<2 mg). Besides that, the content variability of all the generics was higher than that of Cymevene, indicating a poorly controlled filling process. The therapeutic window of ganciclovir is not wide,18 which put forward higher requirements to an appropriate dosage. Lower doses of ganciclovir than expected may lead to an inferior clinical efficacy, while larger doses would result in increased toxicity.
The presence of impurities in the formulation affects the efficacy and safety of the generics seriously. The content of impurities observed in six generic products were generally higher than those in the brand product,indicating that more impurities have been intro- duced during the productive process of some generics. Guanine, the structural backbone of gan- ciclovir, is the largest impurity introducing from synthetic process of the material. Import Drug Registration Standard has put forward specific requirements to its level in the formulation. Our findings suggested that guanine in the Cymevene was too low to be analysed by HPLC, while differ- ent levels guanine could be found in generics.Bacterial endotoxin test revealed that one of the eight generics was identified to be endotoxin conta- minated. The existence of endotoxin in products for injection can lead to pyrogenic responses rang- ing from chills and fever to fatal and irreversible septic shock.19 Any redundant endotoxin residuals have to be strictly avoided to prevent side effects, especially for intravenous medications. Sterility test was also carried out to analyse if microbiological contamination happened in all vials of the prod- ucts. Fortunately, intrinsic microbiological contam- ination could not be found in both generic and brand products, indicating their good security as manufactured injectable products.In addition, the water content in six generics failed to meet the standards to be under 3%. One of the most critical factors in maintaining the qual- ity of lyophilized powders throughout their expected shelf life is well controlled and monitored residual moisture of products. Deterioration of medicines like higher degradation, microorganism growth, polymorph changes, spoilage and poor powder flow properties always resulted from improper levels of moisture in products. Especially for the freeze-dried products, more moisture may cause changes in reconstitution property and the loss of physical structure.
Conclusions
In conclusion, seven of all tested domestically pro- duced generics of ganciclovir for injection failed to reach the in-vitro quality requirements in compari- son with the original brand product Cymevene. As a consequence, discriminations were found in the tests involved in pH measurement, content of API and impurities, endotoxin contamination and residual moisture between generic products and brand product. Above events are of clinical con- cern for patients in need of an effective drug ther- apy, as they directly affect the clinical security and patient tolerance. Ultimately, it is hard to be sim- ply considered that generic products of ganciclovir are essentially equivalent to original brand one, Cymevene. Thus, the substitution of generics to original brand products should be carefully consid- ered before the clinical application.