Accelerated Storage Stability Testing of a Potential Anti-Anthrax Therapeutic, EnvD

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The purpose of stability testing is to determine how the properties of a particular therapeutic vary with time under the influence of specific environmental factors. Information regarding the long-term stability of therapeutics can be extrapolated by performing an accelerated storage stability study. Here, we describe an accelerated storage stability study for the potential anti-anthrax therapeutic, EnvD, a poly-γ-D-glutamic acid (PDGA) depolymerase. Storage conditions were based on those recommended by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). Owing to the high-molecular-weight and associated viscosity of PDGA in solution, loss of enzyme activity on storage can be determined as a reduction in the capacity of the depolymerase to reduce the viscosity of the polymer. This work supported by a Medical Research Council Capacity Building Studentship award.

Materials and Reagents

  1. Poly-γ-D-glutamic acid [prepared in-house; protocol for production can be found in Negus and Taylor (2014)]
  2. Recombinant EnvD enzyme (prepared in-house)
  3. PBS (Thermo Fisher Scientific, catalog number: 12821680 )
  4. Distilled water (prepared in-house)
  5. Compressed air (prepared in-house)
  6. 70% ethanol (see Recipes)


  1. 1.5 ml microcentrifuge tubes (Eppendorf, catalog number: 0030120086 )
  2. Parafilm (Pechiney Plastic Packaging, catalog number: PM996 )
  3. 5 ml sterile plastic syringes (Terumo Medical Corporation, catalog number: SS-05S )
  4. Static incubator (Unitemps, discontinued) (B&T)
  5. Micro-viscometer (Anton Paar GmbH, model: AMVn, catalog number: 1569 )
  6. Glass capillary for micro-viscometer (1.6 mm internal diameter) (Anton Paar GmbH, catalog number: 67605 )
  7. Steel balls (1.5 mm diameter) (Anton Paar GmbH, catalog number: 73109 )
  8. Capillary filling adapater (Anton Paar GmbH, catalog number: 63390 )


  1. Aliquots of freshly isolated EnvD (35 µg total protein) were adjusted to a final volume of 50 µl with sterile PBS in 1.5 ml microcentrifuge tubes and sealed with Parafilm.
  2. Microcentrifuge tubes were maintained at 37 °C over a period of 30 days to simulate long-term storage. The temperature of the incubator was periodically checked to ensure it did not fluctuate by more than ± 2 °C.
  3. Samples of EnvD were periodically (0 days, 1 day, 3 days, 7 days, 15 days, 30 days) removed from the incubator and rapidly combined with 400 µg of lyophilised PDGA substrate suspended in 1 ml of PBS.
  4. Reactions were allowed to proceed for 1 h at 37 °C in a static incubator and were terminated by heating at 95 °C for 10 min. Samples were stored at -20 °C before viscometeric analysis.
  5. Viscosity of PDGA following incubation with stored enzyme preparations was determined using an Anton Paar falling ball microviscometer. Frozen samples were thawed at room temperature and transferred to a glass viscometry capillary containing a solid steel ball using a 5 ml syringe and filling adapter.
  6. Viscosity was determined as the time taken for the ball to fall 25 cm through the sample at an angle of 15° to the horizontal; each automated, timed determination was performed four times.
  7. After each reading the capillary was cleaned with distilled water followed by 70% ethanol. The capillary was then dried with compressed air.

Representative data

Figure 1. Stability of rEnvD during accelerated storage at elevated temperature. Samples of rEnvD were maintained at 37 °C for 30 d. At the time points indicated, aliquots were combined with PDGA (400 μg/ml), incubated for 1 h at 37 °C and the rune time determined. Ball run time (s) was measured in an Anton-Paar AMVn viscometer; the time taken for the ball to fall 25 cm at an angle of 15° to the horizontal was determined. Error bars represent ± 1 SD (n=8).


  1. 70% ethanol
    700 ml ethanol
    300 ml distilled water


This work supported by a Medical Research Council Capacity Building Studentship award.


  1. Kimura, K. and Itoh, Y. (2003). Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate. Appl Environ Microbiol 69(5): 2491-2497.
  2. Negus, D. and Taylor, P. W. (2014). A poly-gamma-(D)-glutamic acid depolymerase that degrades the protective capsule of Bacillus anthracis. Mol Microbiol 91(6): 1136-1147.


稳定性测试的目的是确定特定治疗剂的性质在特定环境因素的影响下如何随时间变化。 关于治疗剂的长期稳定性的信息可以通过进行加速的储存稳定性研究来外推。 在这里,我们描述了潜在的抗炭疽治疗,EnvD,聚-γ-D-谷氨酸(PDGA)解聚酶的加速储存稳定性研究。 储存条件基于国际药品批准技术要求协调会议(ICH)的建议。 由于PDGA在溶液中的高分子量和相关的粘度,储存时酶活性的损失可以被确定为解聚酶降低聚合物粘度的能力的降低。 这项工作由医学研究委员会能力建设学生奖。


  1. 聚-γ-D-谷氨酸[内部制备; 生产协议可以在Negus和Taylor(2014)中找到]
  2. 重组EnvD酶(内部制备)
  3. PBS(Thermo Fisher Scientific,目录号:12821680)
  4. 蒸馏水(内部制备)
  5. 压缩空气(内部制备)
  6. 70%乙醇(见配方)


  1. 1.5ml微量离心管(Eppendorf,目录号:0030120086)
  2. 石蜡膜(Pechiney Plastic Packaging,目录号:PM996)
  3. 5ml无菌塑料注射器(Terumo Medical Corporation,目录号:SS-05S)
  4. 静态培养箱(单位,停止)(B& T)
  5. 微粘度计(Anton Paar GmbH,型号:AMVn,目录号:1569)
  6. 用于微粘度计(1.6mm内径)的玻璃毛细管(Anton Paar GmbH,目录号:67605)
  7. 钢球(直径1.5mm)(Anton Paar GmbH,目录号:73109)
  8. 毛细血管填充适配器(Anton Paar GmbH,目录号:63390)


  1. 在1.5ml微量离心管中用新鲜分离的EnvD(35μg总蛋白)的等分试样用无菌PBS调整至最终体积为50μl,并用Parafilm密封。
  2. 将微量离心管在37℃下维持30天的时间以模拟长期储存。定期检查培养箱的温度,以确保培养箱的温度不会超过±2°C。
  3. 从培养箱中定期取出EnvD样品(0天,1天,3天,7天,15天,30天),并与悬浮在1ml PBS中的400μg冻干的PDGA底物快速合并。
  4. 使反应在37℃下在静态培养箱中进行1小时,并通过在95℃加热10分钟终止。在粘度分析前将样品储存在-20℃
  5. 使用Anton Paar落球微粘度计测定与储存的酶制剂孵育后PDGA的粘度。将冷冻样品在室温下解冻,并使用5ml注射器和填充适配器转移至含有固体钢球的玻璃粘度测定毛细管。
  6. 粘度被确定为球以与水平成15°的角度通过样品下降25cm所花费的时间;每次自动,定时测定四次。
  7. 每次读数后,用蒸馏水,随后用70%乙醇清洁毛细管。 然后用压缩空气干燥毛细管


图1.在升高的温度下加速储存期间rEnvD的稳定性。将rEnvD样品在37℃下保持30天。 在所示的时间点,将等分试样与PDGA(400μg/ml)合并,在37℃下孵育1小时,并确定符合时间。 在Anton-Paar AMVn粘度计中测量球运行时间; 确定球以相对于水平线15°的角度下降25cm所花费的时间。 误差棒表示±1SD(n = 8)。


  1. 70%乙醇
    700 ml乙醇 300 ml蒸馏水




  1. Kimura,K。和Itoh,Y。(2003)。 由噬菌体基因组编码的聚-γ-谷氨酸水解酶的表征:在噬菌体感染的< 用聚γ-谷氨酸封装的枯草芽孢杆菌。 Appl Environ Microbiol 69(5):2491-2497。
  2. Negus,D.和Taylor,P.W。(2014)。 一种聚-γ-(D) - 谷氨酸解聚酶, 芽孢杆菌 炭疽。 91(6):1136-1147。
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引用:Negus, D. and Taylor, P. W. (2014). Accelerated Storage Stability Testing of a Potential Anti-Anthrax Therapeutic, EnvD. Bio-protocol 4(21): e1281. DOI: 10.21769/BioProtoc.1281.

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