Pharmaceutical Technology

1. STAFF DEVELOPMENT PROGRAM
TECHNIQUES OF BIOMEDICAL ENGINEERING
SATURDAY 4th AUG. 2012
FORMULATION OF BIOLOGICALS
Dr. Ashok Hajare
Professor & Head,
Department of Pharmaceutical Technology
Bharati Vidyapeeth College of Pharmacy, Kolhapur

2. Conventional Drugs
Produced by chemical synthesis
Obtained from natural sources, e.g. animal, plant and mineral
Defined structure
Specific physical & chemical characteristics
Micromolecules
Mainly stable

3. Biologicals
Biologicals are delicate drugs
Much larger and more complex
Complex physicochemical structure
Less well-defined
Macromolecule (> 500 kD)
Shear sensitive damaged by unfavorable temperature
Biologicals are useful for treatment of variety of human health disorders,
obtained from biological source and produced through industrial
biotechnology.

4. Biologicals
Species Biomolecule
Animals Serum, vaccines, toxins
Renal monkey tissue Poliomyelitis vaccines
Fluid of chick embryo Mumps and influenza vaccines
Duck embryo Rubella (German measles)
Skin of Bovine calves: inoculation with Smallpox vaccines
vaccine virus
Cell and Tissue cultures vaccines for AIDS and cancer
Genetic engineering: Recombinant DNA Manipulation of spiral DNA chain
and Monoclonal antibody production
Gene splicing: transplantation from higher h-insulin, HGH, hepatitis B
species (human) into lower (bacterium) vaccine, epoetin-alpha, interferon
Monoclonal antibodies Pregnancy testing products

5. FDA Approved Dosage Forms
FDA approved dosage forms in 2010
(international market)
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6. Current Status of Biopharmaceuticals
In 2011, 27 new Molecular Entities
were launched.
India spent >500 crores on R & D,
Employees: more than 46,500
On average, the R&D costs for a
new drug is about 400 Crore.
Global current status of biopharmaceuticals
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Walsh G. Biopharmaceutical benchmarks 2010

7. Storage Conditions Status (Lyophilized Products)
Storage conditions for lyophilized biopharmaceuticals
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8. Current/Future Facts About Biologicals
Biological products are commonly used for chronic health care.
SIGNIFICANT GROWTH
2011 sales: $93B
2015 predicted sales: $175B
1 out of 4 new drugs introduced in US/EU is a biomolecule product
About half of current and new biological products require LYOPHILIZATION
Huge market potential to prepare and deliver biologicals with simple and
cost-effective technologies.

9. Biological Products Approved in India
Human Insulin
Follicle stimulating hormone
Erythropoietin
Human growth hormone
Interferons
Streptokinase
Hepatitis B vaccine
Human interleukin
Granulocyte macrophage
Colony stimulating factor
Blood factors , etc.

10. Erythropoietin
Erythropoietin (EPO or Epoietin), produced primarily by the kidneys.
It is glycoprotein with a molecular weight of approximately 30,000.
It is the principal factor responsible for the regulation of RBC production.
When RBC count comes down (Oxygen level decreases) in the blood
EPO is produced.
EPO can also be produced by rDNA technology.
Should be stored in continuous cold chain to maintain it’s stability.
Widely used in AIDS for development of immunity.
Used in sports to improve endurance.
Used to control anemia associated with chronic kidney disease, cancer
radiation therapy and chemotherapy, malaria.

11. Insulin
Insulin is an important hormone which regulates sugar metabolism.
An inability to produce insulin results in a form of diabetes, this
disease can be treated by daily injections of insulin.
Historically, insulin from pigs or cows is used, but known to produce
immune reactions in some patients.
Recombinant insulin can overcome the problem associated with
immune reactions in some patients.

12. Human Growth Hormone
Secreted by the pituitary gland, and is responsible for normal body
growth and development.
It acts by stimulating protein production in muscle cells and
stimulates the development of bones.
These processes together are responsible for longitudinal growth.
Inadequate production of GH results in short stature, defined as a
below normal height for a given age.

13. Interferons
Class of cytokines effective against viral infections.
Anti viral proteins released by host cells (part of the immune system)
Interfere with viral multiplication.
Host cell specific but not virus specific
Different types of cells in animals produce different interferons.
Types of human interferon: α, β and γ
Α and β - usually produced early in viral infections and γ appears later.
Interferon β - FDA approved - in treatment of cancers and viral diseases.
In treatment of chronic active hepatitis B, chronic hepatitis C, measles,
influenza, multiple sclerosis, etc.

14. Follicle Stimulating Hormone
This hormone is secreted by anterior lobe of the pituitary gland.
It is categorized under the Gonadotropins.
Release of this hormone from anterior pituitary is controlled by
Gonadotropoin-releasing hormone (GnRH) of hypothalamus.
In Females:
Stimulate Follicular growth.
Increases weight of ovaries and enhance production of estrogen.
In Males:
Stimulates testosterone production.
Required for spermatogenesis.
Promotes growth of seminiferous tubule.

15. Biological Product Producers in India
Shantha Biotechnies Pvt. Ltd. Hyderabad
Bharat Biotech International, Hyderabad
Wockhardt, Aurangabad
Reddy’s Labs , Hyderabad
Serum Research Institute Pvt Ltd, Pune
Panacea Biotech, New Delhi
Biological E limited, Hyderabad
Virchow Biotechniques Pvt. Ltd. Hyderabad
Lifeguard Laboratories, Benglore
Strides Arco Lab, Benglore
Benglore Genei, Benglore

16. Production of various Biologicals
From genes
Microorganisms and enzymes
Environmental processes
Fermentation processes
Biotransformation

17. Recombinant DNA and Hybridoma technologies …..
- Production of commercially viable biologicals
- Definite need for skill in formulation
Biomolecule formulation development - challenging area
Critical processing events:
- Purification
- Production and
- Physical and chemical stability
Objective:
Safe, stable and maintains efficacy
Loss of activity during …..
- Processing
- Packaging
- Shipping and
- Long-term storage

18. The Problems With Biologicals
Stability - ‘The tendency to maintain a native (biologically active) conformation’.
Are only marginally stable
X- ray structure analysis of biologicals -
Structure is held together by weak non-covalent forces.
When these forces becomes weak, get broken apart leading to unfolding
and/or inactivation of biologicals.
Chemical instability (Covalent):
Deamidation, Oxidation., Disulfide exchange and Proteolysis.
Physical instability (Non-covalent):
Aggregation and precipitation, Adsorption to surface, and unfolding.
Highly susceptible to both chemical and physical degradation.
- observed in final pack.

19. Mechanisms of Protection
Fundamental understanding of the mechanisms to stabilize
biomolecules.
a) Water Replacement Mechanism
b) Single Amorphous State Immobilization Mechanism
c) Viscosity Mechanism
d) Hydration Protection Mechanism

21. Consideration for Biomolecules Delivery
Source
Physicochemical and storage stability
Physiological barriers
Route of delivery
Pharmacokinetic factors
Formulation type

22. Formulating Biomolecule
Fundamental understanding of the mechanisms to stabilize biomolecules.
CRYO/LYO PROTECTANTS:
Cryoprotectants are preferentially act as structure stabilizers.
Both freezing and dehydration can induce denaturation.
To protect a molecules from freezing (cryoprotection) and from
dehydration (lyoprotection) denaturation, a stabilizer/s is incorporated in
the formulation.

23. BUFFERS
In the development of lyophilized formulations, the choice of
buffer can be critical.
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.
Example: Tris, citrate and histidine buffers.
For stabilizing recombinant factor IX, histidine is found to be the
best.

24. BULKING 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.
Mannitol, sucrose or any other disaccharides are suitable.
Example:
34.5%w/v Sucrose in Rabbit muscle lactate dehydrogenase
formulation.

25. GLASS FORMERS
Disaccharides form an amorphous sugar glass.
Most effective in lyophilization.
Examples - glycerol, xylitol, sorbitol, lactose, mannitol, sucrose, trehalose
and inulin
In comparison with monosaccharide, disaccharides are found to be most
effective.
For example:
Sucrose (30 mM) : Chymotrypsin and growth factors
Glucose and sucrose (1:10): Glucose-6-phosphatedehydrogenase
Trehalose : β-galactosidase, E. coli , etc.

26. TONICITY 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:
BSA (1%) : Rabbit muscle LDH during freezing.
Polyvinyl pyrrolidone : LDH with increased concentrations.
Dextran in sucrose : Actin during lyophilization.

27. METAL IONS
Metal ions can protect some proteins during lyophilization.
Salts and amines have been used as cryoprotectants.
For example:
Zn+ : Insulin protection.
Divalent metal ions (In presence of sugars) : Preserves PFK activity.
Potassium phosphate : Higher recovery of LDH
(sodium cholate and sucrose monolaurate - synergistic effects).

28. SURFACTANTS
Use of surfactants to reduce adsorption and aggregation.
Help in foam formation.
Act as solubilizer: Tween 80, Pluronics, and Brij 35
For example:
Pluronics : Lysozyme, Lasota virus
Tween 80 : Reduce adsorption of calcitonin

29. Techniques
Lyophilization
Spray drying
Vacuum foam drying
Vacuum drying
Precipitations with organic solvents
Air drying
Rotory evaporation
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30. General Limitations
Freezing and moderate low temperatures cause damage to
thermolabile biomolecules.
Process is lengthy and time-consuming.
Not suitable for bulk aseptic production.
Freezing cause damage to biomolecules
Reduces clinical efficacy
Increases risk of adverse effects
Storage facility - cold chain storage and transport

31. Drying
Drying is the final removal of water from material
Commonly - the last stage in a manufacture process.
Methods of drying:
Thermal methods
Non-thermal methods

32. Purpose of Drying
To avoid/eliminate moisture which may lead to corrosion and
decrease the product or drug stability
To improve/keep the good properties of a material, e.g. flowability,
compressibility, etc.
To reduce the cost of transportation of large volume materials
To make the material easy/more suitable for handling

33. Mechanisms of Drying
Spreading the liquid to a thin film
Dispersing the liquid to a spray of small droplets
Freezing the liquid and then drying by sublimation and
desorption

34. Spray Dryer
The spray dryer provides a large
surface area for heat and mass
transfer by atomizing the liquid to
small droplets.
These are sprayed into a stream of
hot air, so that each droplet dries
to a solid particle.
The drying chamber resembles the
cyclone ensuring good circulation
of air, to facilitate heat and mass
transfer, and that dried particles
are separated by the centrifugal
action.

35. Advantages
The droplets are small, giving a large surface area for heat transfer, so
that evaporation is very rapid.
Because evaporation is very rapid, the droplets do not attain a high
temperature, most of the heat being used as latent heat of
vaporization.
The characteristic particle form gives the product a high bulk density
and, in turn, ready solubility.
The powder will have a uniform and controllable particle size.
The product is free-flowing, with almost spherical particles.
Has limited applications in biomolecule processing.

36. Limitations and Applications
Limitations:
The equipment is very bulky, connected to accessories, fans,
heaters,)
That it makes it expensive.
Applications:
Drying in solution or in suspension form.
Useful for drying of thermolabile materials.
Suitable for large quantities solution.
Suitable for both soluble and insoluble substances.
It can produce spherical particles, e.g. dry powder inhalers.

37. Freeze Drying
Freeze drying is a process used to dry extremely heat–sensitive
materials.
It allows the drying, without excessive damage, of proteins, blood
products and even microorganisms, which retain a small but
significant viability.
In this process the initial liquid solution or suspension is frozen, the
pressure above the frozen state is reduced and the water removed by
sublimation.
Three states of matter involved: liquid to solid, then solid to vapour .

38. Freeze Drying Process - Stages
Vacuum application Primary Drying
Freezing stage
stage
Packaging Secondary drying

39. Advantages
Drying takes place at very low temperatures, so the chemical
decomposition, particularly hydrolysis is minimized.
The solution is frozen occupying the same volume as the original
solution, thus , the product is light and porous.
The porous form of the product gives ready solubility.
There is no concentration of solution prior to drying. Hence, salts
do not concentrate and denature proteins, as occurs with other
drying methods.
As the process takes place under high vacuum there is little
contact with air, and oxidation is minimized.

40. Limitations and Applications
Limitations:
The porosity, ready solubility and complete dryness yield a very
hygroscopic product.
The process is very slow and uses complicated plant, which is
very expensive .It is not a general method of drying but limited
to certain types of valuable products.
Applications:
The method is used for products that can not be dried by any
other heat method e.g. antibiotics, blood products, vaccines,
enzyme preparations and microbial cultures.

41. VFD PROCESS
Product Solution
Heat Input, Pressure
Primary Drying
Heat Input, Pressure
Secondary Drying
Heto Lyophilizer
Vacuum sealing
Dry Product
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42. Xerovac Drying
Labile biologicals - extremely sensitive to raised temperature can
be successfully using Xerovac drying.
It is found to be superior method in comparison to freeze drying
Greater energy savings with the Xerovac method
Shorter production cycle (24 h).
Drying by the Xerovac method increases the shelf life, eliminates
the need for preservatives and gives a more stable and less
hazardous product.

43. Comparison of Xerovac and Freeze Drying :
Characteristic Freeze drying Xerovac
Time 48 hours 20-24 hours
Power consumption High Low
Tolerance to power outages Low High
Overall production cost High Low
Residual moisture (3-4%) (1-1.5%)
Storage temperature (-20°C) (4°C or higher)
Heat stability Low 45°C/20 days
(Loss = <1.0 log)
Solubility Moderate Very High (flash)
Biological batch turnover/time Moderate High
Damaging ice crystallization Present Nil

44. Super Critical Fluid Drying
Removes liquid in precisely controlled way.
Dries a substance via high heat and pressure.
It goes around liquid-gas boundary instead of moving through it.
A supercritical fluid is used.
Applications:
Greater energy savings
Much shorter production cycles 24h
Improved production methods
Recently acquired EU accreditation
Large reduction in unit price making its use much cheaper

45. STORAGE
Refrigeration:
Freezing is best for long-term storage.
Low temperature:
Reduces microbial growth and metabolism.
Reduces thermal or spontaneous denaturation.
Reduces adsorption on to the container wall.
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46. PACKAGING
Smooth glass walls best to reduce adsorption or precipitation.
Avoid polystyrene or containers with silanyl or plasticizer
coatings.
Dark, opaque walls reduce chances of oxidation.
Air-tight containers or argon atmosphere reduces air oxidation.
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47. Vacuum Foam Drying (VFD)
Douglas I. Annear stabilized Salmonella ndolo and
Vibrio cholerae by VFD on freeze dried peptone
plugs in the 50’s.
VFD commercialized in 90’s
1. Victor Bronshtein;
Universal Dying Technology, USA
2. Bruce Roser
Quadrant Healthcare, USA (Now VitriLife®)

48. Vacuum Foam Drying Steps
Preconditioning Bubble Formation Foam Formation
Foam Aging

49. Formulation Development by VFD
Ingredients Approx. Quantities Functionality
Biomolecule As per dose Active
Sugar/s 5 – 50% Lyoprotectant and glass formers
PVP 0.5-5% Viscosity enhancer
Pluronics 0.1 – 3.0% Foaming agent
Inorganic water soluble 0.5-5.0% Glass forming agent
phosphate/s
Methyl paraben 0.01-0.15% Preservative
Sodium chloride As required Isotonicity adjusters
SWFI Q. S. Vehicle

50. Product Development Process (VFD)
1. Preformulation studies on biomolecule and excipients
2. Effect of pH and temperature
3. Analytical method development
4. Screening of foaming agent
5. VFD Cycle development
(Temperature, pressure, real time microscopy and DTA/electrical
impedance)
6. Screening of excipients and their concentrations
7. Effect on VFD process parameters
8. Effect of formulation related variables (Fill volume and foam
characteristics)

51. 6. Evaluation of VFD products
(Foamability and foam stability, residual moisture, content analysis
by suitable method-UV/HPLC, FTIR, DSC, XRPD, etc.)
10. Stability studies (ICH Guidelines for Biotechnological/biological
products Q5C)
11. Clinical trials and Pre-clinical studies (Regulatory approvals)
12. Post marketing trials
(Phase IV Clinical Trials - Adverse reaction reporting, Product defect
reposting)
10. Regulatory approvals
11. Distribution

52. Final Comments
Processing biologicals in to the finished products is the most
challenging (and most expensive) of all pharmaceutical
products.
Roughly 50% of all commercial biological products are
lyophilized.
The expertise required for biological formulation development
is vital to the ability of the biopharmaceutical industry to
prepare and market life-saving injectable biological medicines.