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Anhydrobiotic process and excipients for preservation of biomolecules

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Pharmaceutical Technology

Pharmaceutical Technology

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  • 1. Anhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients ForAnhydrobiotic Process And Excipients For Preservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of BiomoleculesPreservation Of Biomolecules 1 A. A. Hajare Professor and Head, Dept. of Pharm. Tech. BHARATI VIDYAPEETH COLLEGE OF PHARMACY, KOLHAPUR ashok.hajare@bharatividyapeeth.edu
  • 2. Recombinant DNA and Hybridoma technologies ….. - Production of commercially viable enzymes, proteins, etc. Result - Definite need for skill in formulation Protein pharmaceuticals development is challenging area BIOMOLECULE THERAPEUTICSBIOMOLECULE THERAPEUTICS 2 challenging area - Production - Purification and - Physical and chemical stability of proteins Loss of activity during ….. - Processing - Packaging - Shipping and - Long-term storage
  • 3. PROTEIN MARKETPROTEIN MARKET Protein pharmaceuticals are (and will be) the most rapidly growing sector in the pharmaceutical repertoire. Most “cures” for difficult diseases (Alzheimer's, cancer, auto-immune diseases, etc.) will probably be found through protein drugs. 3 >210 FDA approved protein drugs >35% are recombinant proteins Protein pharmaceutical sale (2009) : $60 billion Expected to reach (by 2015) : $200 billion/year
  • 4. PROBLEMS IDENTIFIED BY WHOPROBLEMS IDENTIFIED BY WHO All vaccines are unstable and need refrigeration $200 million PA cost of "cold chain" 8 billion injections required p.a. by 2015 Lack of medically trained staff Non-compliance: Patients refuse painful booster injections 12 vaccines per child by 2008 4 12 vaccines per child by 2008 Cross infections - HIV, Hepatitis, septic anemia Solution Required: Completely stable vaccines Single dose units Ready-to-inject
  • 5. SOME BIOMOLECULESSOME BIOMOLECULES 1. Insulin 2. Blood products 3. Herceptin, humulin, alferon 4. Human growth hormone 5. Erythropoietin 6. Antibodies 7. Cytokines 16. Immunomodulators 17. Actimmune 18. Activase 19. BeneFix 20. Betaseron 21. Humulin 22. Novolin 23. Pegademase 5 7. Cytokines 8. Tissue plasminogen activator 9. Urokinase 10. Vaccines 11. Microorganisms 12. Streptokinase 13. Cyclosporine 14. Hormones 15. Immunomodulators 23. Pegademase 24. Epogen 25. Regranex 26. Novoseven 27. Intron-A 28. Neupogen 29. Pulmozyme 30. Infergen
  • 6. BIOMOLECULES DELIVERYBIOMOLECULES DELIVERY Source of protein Physicochemical and storage stability Physiological barriers 6 Route of delivery Pharmacokinetic factors Formulation type
  • 7. ANHYDROBIOSISANHYDROBIOSIS All living organisms require water (75% of most organisms is water) Number of creatures which can survive in a dry state after losing all of their body water e.g. bacteria, fungi, animals and plants. Anhydrobiosis was first recorded by Antoine van Leeuwenhoek, in 1720. A familiar example is baker’s yeast (Saccharomyces cerevisiae) which exist as a dry powder and recovered alive and active by simple rehydration. 7 a dry powder and recovered alive and active by simple rehydration. Organisms are animals : Soil nematodes Plants : Selaginella lepidophylla and Craterostigma plantagineum. All these living things preserve their biological molecules without refrigeration/freezing.
  • 8. 8
  • 9. Anhydrobiosis : Net result of production and accumulation of simple non-reducing sugars (sucrose / trehalose) Many organisms : Uses trehalose Resurrection plant : e.g. Craterostigma uses sucrose 9 R. T. : Many vaccines (e.g. measles), Mcb’s, glucagon, human growth hormones. At 70 C : Enzymes
  • 10. SUGAR GLASS AND STABILITYSUGAR GLASS AND STABILITY Sugar forms a glass on drying in which biomolecules are embedded in solid solution of extremely high viscosity. Therefore, the molecular diffusion required for chemical reaction and 10 degradation is therefore negligible. They are non-reducing and very stable sugars so that the glass matrix cannot participate in chemical reactions with the product, including the Maillard reaction.
  • 11. ANHYDROBIOTIC MECHANISMANHYDROBIOTIC MECHANISM Viscous sugar syrup : during drying forms glass on removal of water Transition - From freely mobile solution in the liquid phase to an immobile solid solution in the glass phase. Stabilization does not occur, if the sugar crystallizes. 11 Molecules are excluded from the sugar crystals.
  • 12. PROTEIN STRUCTUREPROTEIN STRUCTURE Refers to sequence of amino acids & location of disulfide bonds Derived from stearic relations of amino acid residues that are close to one another 12 Refers to overall three dimensional architecture of polypeptide chain The arrangement of two or more polypeptide chains to form a functional protein molecule.
  • 13. In the context of protein structure, the term stability can be defined as ‘The tendency to maintain a native (biologically active) conformation’. Proteins are only marginally stable X- ray structure analysis of water-soluble proteins – Hydrophobic cores of nonpolar amino acids groups surrounded by THE PROBLEMS WITH PROTEINSTHE PROBLEMS WITH PROTEINS 13 Hydrophobic cores of nonpolar amino acids groups surrounded by hydrophilic shell of polar amino acids Structure is held together by weak non-covalent forces. When these forces becomes weak, get broken apart leading to unfolding and inactivation of protein. Highly susceptible to both chemical and physical degradation.
  • 14. Formation or destruction of covalent bonds, within a polypeptide or protein molecule. - Alter the primary structure and impact higher level of its structure. Chemical instability (Covalent): Deamidation, Oxidation. Disulfide exchange and Proteolysis. 14 Deamidation, Oxidation. Disulfide exchange and Proteolysis. Physical instabilitiy (Non-covalent): Aggregation and precipitation, Adsorption to surface, and Protein unfolding. Deamidation and disulphide bond cleavage, may also lead to physical instabilities. Every protein is unique, both physically and chemically, and therefore exhibits unique stability behavior. Physical and chemical instability- May observed in final pack.
  • 15. NONNON--COVALENT PROCESSESCOVALENT PROCESSES 15
  • 16. PROBLEMS WITH BIOMOLECULESPROBLEMS WITH BIOMOLECULES Chemical complexity and marginal stability of higher order structures of therapeutic biomolecules present critical problems in the stability of their formulations. Scientists are working hard to develop a technology that can formulate and deliver life-saving and cheaper biological drugs like vaccines, proteins, enzymes and hormones without refrigeration. About 2 million children die every year from diseases that could be 16 About 2 million children die every year from diseases that could be treated with biomolecule products. About 50% of these life-saving biopharmaceuticals are damaged due to improper storage as well as unavailability of facilities for storing them properly, specifically for temperature effects. To be effective, biomolecules require some mechanism that can maintain their potency and effectiveness at ambient temperature for a sufficiently long time. Biomolecules in a liquid state are stable only for a short period due to molecular movement that may result in degradation.
  • 17. Pharmaceutical products … - Adequate stability over storage periods of several years. - Many biomolecules are unstable in aqueous state at ambient temperature at long-term stability . To attain extended stability at ambient temperature - Molecular movement needs to be arrested by some method that stops 17 - Molecular movement needs to be arrested by some method that stops degradation by transforming liquid into a highly immobile, noncrystalline (amorphous solid) state during storage, called verification. The system below its glass transition temperature (Tg) is stable due to immobilization of the reactive entity in a solid glass-like system.
  • 18. pH Ionic strength Oxidants Free radicals DENATURANTSDENATURANTS 18 Free radicals Heat Mechanical stress: Shear, Shaking Pressure
  • 19. PROTECTANTSPROTECTANTS Formulating biomolecule: Fundamental understanding of the mechanisms to stabilize proteins. Cryo and Lyo protection: Nature protects life from freezing by accumulating selected compounds to high concentration within organisms. 19 Cryoprotectants are preferentially excluded from surface of proteins and act as structure stabilizers. Both freezing and dehydration can induce protein denaturation. To protect a protein from freezing (cryoprotection) and from dehydration (lyoprotection) denaturation, a protein stabilizer/s is incorporated in the formulation.
  • 20. BUFFERSBUFFERS In the development of lyophilized formulations, the choice of buffer can be critical. Phosphate buffers particularly phosphate; undergo drastic pH changes during freezing. A good approach is to use low concentration of a buffer that undergoes minimal pH changes during freezing such as Tris, citrate and histidine buffers. 20 For example: 8.4 µg/ml of ox liver catalase in 10 mM phosphate buffer (pH 7.0) freezing at - 15ºC to -75ºC retained 80% of activity. About 0.5 mg/ml of LDH in 0.1 M NaCl and 10mM phosphate buffer (pH 7.5) retained 76% of the activity13. For stabilizing recombinant factor IX, histidine is found to be the best.
  • 21. BULKING AGENTSBULKING AGENTS Bulking agents are added to provide bulk to the formulation. Important at very low concentrations of biomolecules. Crystalline bulking agents produce an elegant cake structure with good mechanical properties. 21 Mannitol, sucrose or any other disaccharides are suitable. For example, Sucrose (34.5% w/v) : Rabbit muscle lactate dehydrogenase.
  • 22. SUGARSSUGARS Disaccharides form an amorphous sugar glass. Most effective in lyophilization. Sugars like glycerol, xylitol, sorbitol, lactose, mannitol, sucrose, trehalose and inulin – used as cryoprotectant and lyoprotectant. In comparison with monosaccharide, disaccharides are found to be most effective. 22 For example: Sucrose (30 mM) : Chymotrypsin and growth factors Glucose and sucrose (1:10) : Glucose-6-phosphatedehydrogenase Trehalose : β-galactosidase, S- adenosyl - L- methionine, E. coli and B.Thuringienesis.
  • 23. TONICITY ADJUSTERSTONICITY ADJUSTERS Needed either for stability or for route of administration. Mannitol, sucrose, glycine, glycerol, sodium chloride, polymers, etc. Increased concentrations showed increased activity. For example: 23 For example: BSA (1%) : Rabbit muscle LDH during freezing. Polyvinyl pyrrolidone : LDH with increased concentrations. Dextran in sucrose : Actin during lyophilization.
  • 24. METAL IONSMETAL IONS Metal ions can protect some proteins during lyophilization. Salts and amines have been used as cryoprotectants. For example: Zn+ : Insulin protection. 24 Divalent metal ions (In presence of sugars) : Preserves PFK activity. Potassium phosphate : Higher recovery of LDH (sodium cholate and sucrose monolaurate - synergistic effects).
  • 25. SURFACTANTSSURFACTANTS Use of surfactants to reduce adsorption and aggregation. Help in foam formation. Act as solubilisers Tween 80, Pluronic F-68, and Brij 35 25 For example: Pluronics : Lysozyme, Lasota virus : Reduce adsorption of calcitonin
  • 26. BIOMOLECULE PROTECTIONBIOMOLECULE PROTECTION Stresses in solutions - heating, hydrolysis, agitation, freezing, pH changes and exposure to denaturants. The net result - inactivation or aggregation - less clinical efficacy - high risk of adverse side effects The practical solution - remove the water. 26 To develop formulation - specific conditions and proper stabilizing additives Uniqueness of protein - responsible for specific routes of chemical and physical degradation during lyophilization and storage. Difficult to predict degradation pathway by simply designing formulation.
  • 27. MECHANISMS OF PROTECTIONMECHANISMS OF PROTECTION Lyophilization / Rehydration: a) Thermodynamic Mechanism b) Protein Cryoprotectant Complex Mechanism c) Diffusion Restriction Mechanism 27 Drying: a) Water Replacement Mechanism b) Single Amorphous State Immobilization Mechanism c) Viscosity Mechanism d) Hydration Protection Mechanism
  • 28. Techniques: Spray drying, freeze drying or lyophilisation, freeze thawing, precipitations with organic solvents, air drying and rotors evaporation Major limitations: Freezing and moderate low temperatures cause damage to TECHNIQUES AND LIMITATIONSTECHNIQUES AND LIMITATIONS 28 Freezing and moderate low temperatures cause damage to thermolabile biomolecules, reducing their clinical efficacy and increasing the risk of adverse effects. Process is lengthy and time-consuming. If formulated successfully, storage facility such as cold chain storage transport is a must to maintain stability. Not suitable for bulk aseptic production.
  • 29. Protein solution atomised and particles dried in seconds in an air stream. Major advantage - Spherical particles produced - Good flow properties with control over particle size - Very useful for design of non-parenteral dosage forms SPRAYSPRAY--DRYINGDRYING 29 Use - Materials that can withstand high temperatures during drying Unsuitable: Damage to sensitive biologicals and pharmaceuticals High temperature requirement
  • 30. During cryopreservation by freezing - Damage with formation of ice crystals During preservation by cryovitrification, the specimen are subjected to toxic effects of concentrated vitrification CRYOPRESERVATIONCRYOPRESERVATION 30 subjected to toxic effects of concentrated vitrification solutions Damage caused during freezing and cryopreservation limits survival or activity yielded after preservation.
  • 31. FREEZEFREEZE -- DRYINGDRYING Cost-effective, and produces chemically stable and active protein. Best for long term storage. Removes a considerable amount of water. 31 Freezing of specimens before lyophilization and equilibrium of specimens in partially frozen state can be very damaging. Cryoprotectants are used to prevent damage. After lyophilization needs refrigerated storage conditions.
  • 32. FREEZEFREEZE--DRYING PROCESSDRYING PROCESS Biological items are first frozen in container. Place under strong vacuum. Solvent sublimates leaving only solid at 32 Solvent sublimates leaving only solid at intermediately low temperatures (above 50ºC) Reduces moisture content to <0.1%
  • 33. STORAGESTORAGE Refrigeration: Freezing is best for long-term storage. Low temperature: Reduces microbial growth and metabolism. 33 Reduces microbial growth and metabolism. Reduces thermal or spontaneous denaturation. Reduces adsorption on to the container wall.
  • 34. Smooth glass walls best to reduce adsorption or precipitation. Avoid polystyrene or containers with silanyl or plasticizer coatings. PACKAGINGPACKAGING 34 Dark, opaque walls reduce chances of oxidation. Air-tight containers or argon atmosphere reduces air oxidation.
  • 35. VACUUM FOAM DRYING (VFDVACUUM FOAM DRYING (VFD)) 35 ‘Scalable long-term shelf preservation technique for sensitive biologicals’
  • 36. EVAPORATIVE Vs FREEZE DRYINGEVAPORATIVE Vs FREEZE DRYING Very few scientists working… Annear, Bronshtein, Roser, Pisal, etc. Preservation of biological fluids and components, proteins, enzymes and micro-organisms Evaporative drying for long periods at ambient temperature without significant loss of activity. 36 significant loss of activity. Observations: 1. Stability is better than that of freeze-dried samples. 2. Dehydrated solutions with protectants are viscous. 3. The process is under industrial scale up stage.
  • 37. FOAM FORMATIONFOAM FORMATION For the last 50 years… Freeze drying has been the best method for stabilization due to belief that low temperatures cause minimum damage. Preservation by foam formation (PFF) is a new technology… - Proposed by Bronshtein in 1996. 37 According to Bronshtein, this belief in low-temperature drying with minimum damage is not correct. Before Bronshtein’s invention of foam formation, no scalable technology had been proposed to preserve thermolabile biomolecules at ambient temperature.
  • 38. Preserved bacteria in a dried state. Claim: Viscous solutions and biological liquids can be dried by forming foam by applying a vacuum. ANNEAR et. al. 38 He used this FFP for a only small volume of sample. FFP was not used until recently, because it was considered to be a process that damages biologicals.
  • 39. BRONSHTEIN et. al.BRONSHTEIN et. al.BRONSHTEIN et. al.BRONSHTEIN et. al. First to report that biologicals could be effectively stabilized by foam drying. Claim: PFF has been used successfully to dry various volumes of biological liquids from 1-100,000ml. 39 liquids from 1-100,000ml. The only limitation of this technology is that the volume of liquid to be dried must not be more than 20-25% of the container volume, because the sample expands during foam formation. The time required for this process is much shorter than other processes due to intensive boiling.
  • 40. FOAM FORMATION PROCESSFOAM FORMATION PROCESS In this process, the biological solutions or suspensions are first transformed into mechanically stable dry foams by boiling them under vacuum at ambient temperature above freezing point but significantly below 100ºC (primary drying). Samples are then subjected to stability drying at an elevated temperature to increase glass transition temperature (Tg). 40 g Activity yield after the rehydration of the foam-dried sample is achieved by proper selection of protectants (sugars like sucrose and trehalose), which are dissolved in the suspension before processing. Proper selection and use of vacuum, as well as temperature protocols during drying, help to produce elegant and therapeutically active products that remain stable at an ambient storage temperature.
  • 41. Suspension containing a biologically active agent is dehydrated or concentrated by evaporation to high vacuum of pressure higher than 7.6 Torr. Then pressure adjusted in between 0- 7.6 Torr. This is sufficient to cause boiling and this lead to mechanically stable dried foam during boiling. 41 Secondary drying is carried out by applying vacuum or dry air to form stable at elevated temperature Surfactant is added to enhance foam stability during secondary drying. Protectant is selected from a group consisting of sugar, carbohydrate, polysaccharide, polymer, peptide, protein or their mixture.
  • 42. ADVANTAGES OF PFFADVANTAGES OF PFF Scalable and turbulent process with efficient preservation capability. Stability of sensitive biologicals at room temperatures. Lends itself as an aseptic process due to higher vapor pressure above the sample during PFF, leading to less surface area exposure and less exposure time. Does not require freezing of sample before drying, therefore more efficient, gentle and less damaging. Less time consuming and more energy efficient. More scalable process compared to freeze drying, which has limitation of cake height in container. 42 height in container. Allows high ambient temperature stabilization with minimum loss of activity during drying and storage. Offers the potential to deliver biomolecules outside the cold chain storage. High production yields and Long shelf life. Materials are shipped at ambient temperatures, eliminating refrigerated or frozen storage & spoilage due to handling & power failures. Distribution in areas where refrigeration and freezing facilities are not available or inadequate (under developed countries)
  • 43. APPLICATIONS OF VFDAPPLICATIONS OF VFD PFF has been used successfully for stabilisation of thermolabile enzymes and pharmaceuticals: Amphotericin, urokinase, luciferase, ß-galactosidase, lactate dehydrogenase, isocitric dehydrogenase, Isocitrate dehydrogenase, erythopoeitin, lysozyme and icenucleating proteins at ambient or higher temperature. Live viruses: Lasota, herpesvividae, paramyxovividae, flaviviridae, 43 Lasota, herpesvividae, paramyxovividae, flaviviridae, parvoviridae and retroviruses can also be stabilised by using this vacuum foam drying technique. Gram-negative bacteria : E. coli and B. bronchiseptica Gram-positive bacteria : Lactobacillus acidophilus and Lactococcus lactis subspecies. Thermolabile antibiotic such as doxorubicin.
  • 44. India - Formulation and preservation research is limited. Development of stable pharmaceuticals - Much slower pace. Limitations of current technologies- 1. Retain less biological activity 2. Require long processing time WHY VFD?WHY VFD? 44 2. Require long processing time 3. Produce short shelf life products 4. Cold chain storage and transport systems 5. Induces stresses that denatures proteins.
  • 45. VFD COMPARED TO LYOPHILIZATIONVFD COMPARED TO LYOPHILIZATION CONSIDERATION VFD TECHNOLOGY LYOPHILIZATION EFFICIENCY - Boiling materials under vacuum at temperature above 0°C. - Very efficient. - Reduce spoilage due to handling and power failures - Inefficient - Time consuming - comparatively less efficient CYCLE TIME 24 Hours 2 - 10 days SCALABILITY - Formation of stable foam to form thin films. - Allow efficient removal of water at broad range of volumes. - Drying rate is limited by cake-height in each container. - Scalability is achieved by - At room as well as at higher temperatures. - Scalability is achieved by using more containers. YIELD - Water evaporates at temperatures above samples freezing point - Eliminates damage due to freezing. - High production yields - The need to freeze before sublimation of water can damage the material. - Lead to lower yields. TEMPERATURE STABILITY - Combination of protective fillers and dehydration process allows high temperature stability. - Preserves broad range of materials at up to 50°C and can be shipped at room temperature. - Long shelf life - Most of freeze dried samples are stable under refrigeration. - In some cases at room temperature - Short shelf life 45
  • 46. FOAM FORMATION EQUIPMENTFOAM FORMATION EQUIPMENT At the present time no special industrial equipment has been designed and is available for the bulk production of powders or market-ready vials by the vacuum foam drying technique. Researchers have claimed that with a few modifications to the controls and process cycle programming software, commercially available freeze dryers could be modified for PFF in glass vials. 46 available freeze dryers could be modified for PFF in glass vials. The main requirement is simultaneous control of vacuum and temperature during foam drying. Thus, the pharmaceutical and other industries are suffering from an absence of effective drying equipment that produces bulk products stable at ambient temperature.
  • 47. Optimization of temperature and pressure cycles: 1. Vacuum concentration 2. Stability drying and PROCESS DEVELOPMENTPROCESS DEVELOPMENT 47 3. Rapid cooling for glassy matrix.
  • 48. 120 140 160 180 200 Foamheight(mm) 0.5%- F108 1%-F108 3%-F108 0.5%-F68 1%-F68 3%-F 68 0.5%-F87 1%-F87 3%-F87 VFD OF LASOTAVFD OF LASOTA : SCREENING OF FOAMING AGENTSCREENING OF FOAMING AGENT 48 0 20 40 60 80 100 0 20 40 60 80 100 120 140 Tim e(m in) Foamheight(mm)
  • 49. VFD CYCLE OPTMIZATIONVFD CYCLE OPTMIZATION STEP CYCLE 1 CYCLE 2 CYCLE 3 Temp (°C) Vacuum (mT) Time (Min) Temp (°C) Vacuum (mT) Time (Min) Temp (°C) Vacuum (mT) Time (Min) 1 8 4000 120 18 4000 120 -10 - 15 2 10 4000 120 20 4000 120 10 1200 60 3 10 1500 120 20 1500 120 15 1000 60 4 12 1500 120 22 1500 120 20 800 120 5 14 1500 120 24 1500 120 22 600 120 6 16 1200 60 26 1200 60 24 200 120 49 6 16 1200 60 26 1200 60 24 200 120 7 18 1200 60 28 1200 60 26 100 120 8 20 400 120 30 400 120 28 25 120 9 22 200 60 32 200 60 28 25 120 10 24 200 60 34 200 60 28 25 120 11 26 100 120 36 100 120 28 25 120 12 28 25 120 38 25 120 30 25 240 13 30 25 120 40 25 120 40 25 120 14 40 25 120 26 25 120 26 25 120 15 26 25 120 - - - - - -
  • 50. CURRENT STATUS OF VFDCURRENT STATUS OF VFD It is a new processing technique. Yet not much exploited but has better potential. The concept is under investigation for larger scale. 50 The products are stable at room temperatures.
  • 51. LIMITATIONSLIMITATIONS Although foam formation is an old invention, preservation by foam formation under vacuum and controlled temperature is a new technology in the embryonic stage, being used for only a few pharmaceutical applications and needs some improvement. Elimination of uncontrolled eruptions and spitting out of material from vials or containers during boiling are the improvements required in this technology. 51 technology. More parameters must be studied for its comparison with freeze drying and other known and newly developed drying processes.
  • 52. Preservation by foam formation may be a substitute to freeze drying or lyophilisation and will stimulate development of new processes and equipment for preservation of thermolabile biologicals in a dry state. COMMENTSCOMMENTS