DEVELOPMENT AND OPTIMIZATION OF LYOPHILIZATION CYCLE OF THE MODEL ANTIBIOTIC

1. DEVELOPMENT AND OPTIMIZATION OF LYOPHILIZATION CYCLE OF THE MODEL ANTIBIOTIC Name of student : Lakshmi T.P. Specialization : Pharmaceutics Name of academic guide : Mrs. Deepa Karthikeyan Name of the Industry : Strides Arcolabs Place : Bangalore Name of Industrial coordinator : Name of Industrial guide :

2. NEED OF STUDY:- The model antibiotic under study being unstable in solution form is converted to a stable dry powder form by Lyophilization technique.

3. OBJECTIVE OF STUDY: To develop and optimize the lyophilization cycle of the model antibiotic for parenteral administration and study the impact of lyophilization cycle on the final product.

4. GOALS: • Study the factors that affect lyophilization. • To develop the lyophilization cycle. • Optimization of lyophilization cycle. • Evaluation of lyophilized product.

5. LYOPHILIZATION-(FREEZE-DRYING): • DEFINITION :- The creation of a stable preparation of a biological substance by rapid freezing and dehydration of the frozen product under high vacuum1.

6. Advantages • Product is stored in dry state-few stability problems. • Product is dried without elevated temperatures. • Good for oxygen and/or air-sensitive drugs. • Rapid reconstitution time. • Constituents of the dried material remain homogenously dispersed. • Product is processed in liquid form. • Sterility of product can be achieved and maintained

7. Disadvantages • Volatile compounds may be removed by high vacuum. • Single most expensive unit operation. • Stability problems associated with individual drugs. • Some issues associated with sterilization and sterility assurance of the dryer chamber and aseptic loading of vials into the chamber. • High energy costs (2-3times more than other methods). • Long process time.

8. The freeze drying process: • Four stages in drying process: • Freezing • Primary drying • Secondary drying.

9. Freezing : • The materials are cooled below its triple point, the lowest temperature at which solids and liquids phases of the material coexist. • This ensures sublimation rather than melting. • Freezing at ordinary pressure.

10. Primary drying : • The pressure is lowered, and enough heat is supplied to the material for the solvent (ice) to sublimate. • The temperature of the product must remain slightly below its critical temperature. • About 95% of solvent in the material gets sublimed. • This phase may be slow because, if too much heat is added, the material’s structure could be altered.

11. Secondary drying : • To remove bound water from the solutes to a level that assures long term stability of the product. • The temperature is raised than primary drying phase and can be above 0 °C (upto 40°C). • The pressure is lowered in this phase to encourage desorption. • At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%.

12. Applications : • Pharmaceutical and biotechnology : • To increase the shelf life of products, such as vaccines and other injectables. • The material can be easily stored, shipped, and later reconstituted to its original form for injection. • In bacteriology freeze-drying is used to conserve special strains. • Food Industry : • Freeze-drying is used to preserve food • Make it very lightweight. Eg:-freeze-dried ice cream , an example of astronaut food, some instant coffee .

13. Technological industry • Some products of chemical synthesis are easier to dissolve in water for subsequent use. • In bioseparations,it can be used as a late-stage purification procedure. • It is capable of concentrating substances with low molecular weights that are too small to be removed by a filtration membrane. • Freeze-drying is reserved for materials that are heat-sensitive, such as proteins, enzymes, microorganisms, and blood plasma.

15. Factors that affect lyophilization • Glass transition temperature (Tg% ): • Collapse temperature (Tcol), • Crystallization temperature (Tcry), • Eutectic temperature (Teut), and • Devitrification temperature (Tdev), These temperatures are mostly determined by thermal analysis such as: • DSC, electrical resistance measurements • Freeze drying microscopy.

16. Glass transition temperature (Tg% ) • Ice formation during cooling of a solution concentrates all solutes. • Solute concentration eventually changes the solution from a viscous liquid to a brittle glass, which contains about 20–50% water • The temperature of this reversible transition for the maximally freeze-concentrated solution is termed glass transition temperature, Tg% .

17. The collapse temperature (Tcol) The collapse temperature (Tcol) is the temperature at which the interstitial water in the frozen matrix becomes significantly mobile. Tcol is closely related to Tg% . Tcol has been considered to be equivalent to Tg of an amorphous system or to the eutectic melting temperature of a crystalline system

18. Crystallization temperature (Tcry) When the temperature of an aqueous formulation drops below 0°C, water usually crystallizes out first. The crystalline component, which usually has the least solubility in the formulation, may crystallize out. This temperature is termed crystallization temperature.

19. Eutectic crystallization: melting temperature (Teut) When the temperature of an aqueous formulation further decreases after crystallization of the least soluble component, this component and water crystallize out at the same time as a mixture. This temperature is termed eutectic crystallization:

20. Theoretical phase diagram showing ice formation, solute crystallization, eutectic point, and glass transition during freezing.

21. Freeze drying cycle optimization Primary Drying Conservative drying conditions: product temperature well below critical formulation temperature to avoid collapse High product temperature to reduce time cycle Optimized cycle 21 Only an optimized cycle meets the demands of time and quality

22. Characteristics of lyophilized products • Sufficiently dry • Sufficiently porous • Sufficient strength • Uniform colour • Sterile • Free of pyrogens • Free of particulates • Chemically stable

23. Critical formulation temperature: • Eutectic temperature (Teut) for crystalline substances (e.g.mannitol) Above this temperature the solid product begins to melt • Glass transition temperature of the maximally freeze concentrated solute (Tg’) and collapse temperature (Tc) for amorphous substances (e.g. sucrose) Above this temperature the viscosity of the glassy solid decreases, product morphology changes Product temperature at the ice sublimation interface must be maintained below the critical formulation temperature throughout the primary drying step

24. COLLAPSE: With beginning sublimation the ice-vapor interface moves on and leaves a porous, sponge like dried product matrix Drying above Tc decreases viscosity of the dried structure, the product matrix flows into the pores (viscous flow) This structural loss is visible in the microscope Negative effects on product quality attributes: Lack of elegance Elevated residual moisture content Extended/uncompleted reconstitution Reduced storage stability Instability of the active pharmaceutical ingredient (API)

25. PROCESS DEVELOPMENT CHALLENGES • Development of an optimized lyophilisation cycle. • Understanding the characteristics of the product and the lyophiliser. • Heat transfer characteristics of the vial system in use. • Shortening the lyophilisation cycle development process to produce an optimized lyophilisation cycle can • Increase efficiency. • Accelerate development time. • Thus reduce time to market and save valuable product.

26. THE LYOPHILIZATION PROCESS GENERALLY INCLUDES THE FOLLOWING STEPS: Dissolving the drug and excipients in a suitable solvent, Sterilizing the bulk solution by passing it through a 0.22 micron bacteria-retentive filter. Filling into individual sterile containers and partially stoppering the containers under aseptic conditions. Transporting the partially stoppered containers to the lyophilizer and loading into the chamber under aseptic conditions. Freezing the solution by placing the partially stoppered containers on cooled shelves in a freeze-drying chamber or pre-freezing in another chamber. Applying a vacuum to the chamber and heating the shelves in order to evaporate the water from the frozen state. Complete stoppering of the vials usually by hydraulic or screw rod stoppering mechanisms installed in the lyophilizers.

27. Characteristics of the drug under study Description: The model antibiotic is a mixture of related cyclic polypeptides produced by organisms of the licheniformis group of Bacillus subtilis var Tracy. As a toxic and difficult-to-use antibiotic, it doesn’t work well orally. However, it is very effective parentraly and topically.

28. Synthesis : Synthesised via the so-called nonribosomal peptide synthetases (NRPSs), which means that ribosomes are not involved in its synthesis. • Molecular weight : 1422.693 • Chemical formula : C66H103N17O16S • Category : Anti-Bacterial Agents Anti-Infective Agents

29. pharmacology • Indications : • Antibacterial action in vitro against a variety of gram-positive and a few gram-negative organisms. • In the treatment of infants with pneumonia and empyema, caused by staphylococci. • Topical treatment for a variety of localized skin and eye infections, as well as for the prevention of wound infections. • Inhibitor of proteases and other enzymes. • Widely used in operating rooms to soak implants, irrigate compound fractures, and apply to surgical incisions6

30. Mechanism of action: • The model antibiotic intereferes with the dephosphorylation of the 55-carbon, biphosphate lipid transport molecule C55-isoprenyl pyrophosphate (undecaprenyl pyrophosphate), which carries the building blocks of the peptidoglycan bacterial cell wall outside the inner membrane for construction. • It binds divalent transition metal ions (Mn(II), Co(II), Ni(II), Cu(II), and Zn(II)) which binds and oxidatively cleave DNA

31. PHARMACOKINETICS Absorption: • Intramuscular injection - rapid and complete. • Gastrointestinal tract following oral administration - not appreciable. • Topical application - negligible. Distribution: • Widely distributed in all body organs • Demonstrable in ascitic and pleural fluids after intramuscular injection. Excretion: • Excreted slowly by glomerular filtration.

32. MATERIALS AND METHODS: • SOURCE OF DATA • 1. Review of literature from • a. Journals such as:- • European Journal of Pharmaceutical Sciences • PDA Journal of Pharmaceutical Science and Technology. • European Pharmaceutical Review • International Journal of Heat and Mass Transfer • International Journal of Pharmaceutics • Chemical Engineering Science

33. b. Research literature data bases such as: • Science Direct • Pubmed • c. Standard books such as • Analytical Profiles of Drug Substances • Martindale- The Complete Drug Reference • Remington: The Science and Practice of Pharmacy • d. Internet browsing • www.google.com • www.wikipedia.org

34. METHOD OF COLLECTION OF DATA: From literature Laboratory based studies. Preformulation studies. Formulation of injectable dosage form of the model antibiotic. Perform the hold time studies. Perform tubing compatibility studies. Optimization of lyophilization cycle. Evaluation of lyophilized product for reconstitution time, Loss on drying, pH of reconstituted solution, particulate matter, assay.

35. REVIEW OF LITERATURE: Wang D et al., formulated Polymyxin E sulfate, a hydrophilic drug with high tissue toxicity into a stable liposome to reduce its in vivo toxicity. He used freeze-drying (lyophilization) technique to prepare the proliposome. It was found that the stability and encapsulation efficiency of the liposome could be improved by the freezing-thawing process. Di Tommaso C et al., studied the applicability of lyophilization to polymeric micelles based on methoxy poly(ethylene glycol)-poly(hexyl-lactide) (MPEG-hexPLA). Freeze-thawing tests were carried out to determine the protective effect of various excipients on the freezing step. Mannitol, trehalose, glucose and sucrose showed the best effectiveness in micelle protection. This study showed that the MPEG-hexPLA micelles can be efficiently lyophilised and this process can be usefully applied to increase the pharmaceutical stability of these pharmaceutical micelle formulations.

36. M. Brülls and A. Rasmuson studied the heat transfer in vial lyophilization experimentally and theoretically. The factors studied were bottom curvature of the vial, chamber pressure, fill volume, position on the shelf and the state of the water. The effect of the curvature of the vial bottom, the heat accumulation in the glass vial and the heat transfer to the sidewalls of vials in the corner of the shelf all contributed to a significant radial influence on the heat transfer.

37. REFERENCES: Wang D et.,al . Polymyxin E sulfate-loaded liposome for intravenous use: preparation, lyophilization, and toxicity assessment in vivo. PDA Journal of Pharmaceutical Science Technology. 2009 Mar-Apr;63(2):159-67. Di Tommaso C et al., Investigations on the lyophilization of MPEG-hexPLA micelle based pharmaceutical formulations. Eur J Pharm Sci.- 2010 Apr16; 40(1):38-47. Klaus Florey. Analytical Profiles of Drug Substances. Volume. 2005; 9: Pages 1-40 Sean C. Sweetman. Martindale- The Complete Drug Reference. 36th edition; volume 1: Page no. 162. M. Brülls and A. Rasmuson.Heat transfer in vial lyophilization. International Journal of Pharmaceutics, 2002 October 10 th;Volume 246, Issues 1-2: Pages 1-16 Susanne Hibler and Dr. Henning Gieseler. Primary packaging materials for pharmaceutical freeze-drying: Moulded vs. serum tubing vials. European Pharmaceutical Review- 2010 August 19th ,Issue 4 2010.

38. THANKS