FORMULATION OF BIOLOGICALS

STAFF DEVELOPMENT PROGRAM TECHNIQUES OF BIOMEDICAL ENGINEERING

SATURDAY 4th AUG. 2012

Dr. Ashok HajareProfessor & Head,

Department of Pharmaceutical Technology

Bharati Vidyapeeth College of Pharmacy, Kolhapur

� Produced by chemical synthesis

� Obtained from natural sources, e.g. animal, plant and mineral

� Defined structure

� Specific physical & chemical characteristics

Conventional Drugs

� Specific physical & chemical characteristics

� Micromolecules

� Mainly stable

� Biologicals are delicate drugs

� Much larger and more complex

� Complex physicochemical structure

� Less well-defined

� Macromolecule (> 500 kD)

Biologicals

� 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.

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

vaccine virus

Smallpox vaccines

� Cell and Tissue cultures vaccines for AIDS and cancer

� Genetic engineering: Recombinant DNA

and Monoclonal antibody production

Manipulation of spiral DNA chain

� Gene splicing: transplantation from higher

species (human) into lower (bacterium)

h-insulin, HGH, hepatitis B

vaccine, epoetin-alpha, interferon

� Monoclonal antibodies Pregnancy testing products

FDA Approved Dosage Forms

5

FDA approved dosage forms in 2010

(international market)

Current Status of Biopharmaceuticals

� In 2011, 27 new Molecular Entities

were launched.

� India spent >500 crores on R & D,

� Employees: more than 46,500

6Walsh G. Biopharmaceutical benchmarks 2010

� Employees: more than 46,500

� On average, the R&D costs for a

new drug is about 400 Crore.

Global current status of biopharmaceuticals

Storage Conditions Status (Lyophilized Products)

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Storage conditions for lyophilized biopharmaceuticals

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

Current/Future Facts About Biologicals

� 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.

Biological Products Approved in India

� Human Insulin

� Follicle stimulating hormone

� Erythropoietin

� Human growth hormone

� Interferons

� Streptokinase� Streptokinase

� Hepatitis B vaccine

� Human interleukin

� Granulocyte macrophage

� Colony stimulating factor

� Blood factors , etc.

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.� 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.

Insulin

� Insulin is an important hormone which regulates sugar metabolism.

� An inability to produce insulin results in a form of diabetes, thisdisease can be treated by daily injections of insulin.

� Historically, insulin from pigs or cows is used, but known to produceimmune reactions in some patients.immune reactions in some patients.

� Recombinant insulin can overcome the problem associated withimmune reactions in some patients.

Human Growth Hormone

� Secreted by the pituitary gland, and is responsible for normal bodygrowth and development.

� It acts by stimulating protein production in muscle cells andstimulates the development of bones.

� These processes together are responsible for longitudinal growth.� These processes together are responsible for longitudinal growth.

� Inadequate production of GH results in short stature, defined as abelow normal height for a given age.

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� 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.

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.� 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.

� 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 Product Producers in India

� Panacea Biotech, New Delhi

� Biological E limited, Hyderabad

� Virchow Biotechniques Pvt. Ltd. Hyderabad

� Lifeguard Laboratories, Benglore

� Strides Arco Lab, Benglore

� Benglore Genei, Benglore

Production of various Biologicals

� From genes

� Microorganisms and enzymes

� Environmental processes

� Fermentation processes� Fermentation processes

� Biotransformation

� Recombinant DNA and Hybridoma technologies ….. - Production of commercially viable biologicals

- Definite need for skill in formulation

� Biomolecule formulation development - challenging areaCritical processing events:

- Purification

- Production and

- Physical and chemical stability- Physical and chemical stability

Objective:

Safe, stable and maintains efficacy

� Loss of activity during …..- Processing

- Packaging

- Shipping and

- Long-term storage

� 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.

The Problems With 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.

� Fundamental understanding of the mechanisms to stabilize

biomolecules.

a) Water Replacement Mechanism

b) Single Amorphous State Immobilization Mechanism

Mechanisms of Protection

c) Viscosity Mechanism

d) Hydration Protection Mechanism

� Source

� Physicochemical and storage stability

� Physiological barriers

Consideration for Biomolecules Delivery

� Physiological barriers

� Route of delivery

� Pharmacokinetic factors

� Formulation type

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.

Formulating Biomolecule

� 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.

BUFFERSBUFFERS

� 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.undergoes minimal pH changes during freezing.

Example: Tris, citrate and histidine buffers.

� For stabilizing recombinant factor IX, histidine is found to be the

best.

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.

GLASS FORMERSGLASS 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.effective.

For example:

Sucrose (30 mM) : Chymotrypsin and growth factors

Glucose and sucrose (1:10): Glucose-6-phosphatedehydrogenase

Trehalose : β-galactosidase, E. coli , etc.

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:For example:

BSA (1%) : Rabbit muscle LDH during freezing.

Polyvinyl pyrrolidone : LDH with increased concentrations.

Dextran in sucrose : Actin during lyophilization.

METAL IONSMETAL 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).

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

Techniques

� Lyophilization

� Spray drying

� Vacuum foam drying

� Vacuum drying

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� Vacuum drying

� Precipitations with organic solvents

� Air drying

� Rotory evaporation

� 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

General Limitations

� Reduces clinical efficacy

� Increases risk of adverse effects

� Storage facility - cold chain storage and transport

� Drying is the final removal of water from material

� Commonly - the last stage in a manufacture process.

� Methods of drying:

Drying

� Methods of drying:

� Thermal methods

� Non-thermal methods

� 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.

Purpose of Drying

compressibility, etc.

� To reduce the cost of transportation of large volume materials

� To make the material easy/more suitable for handling

� 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

Mechanisms of Drying

� Freezing the liquid and then drying by sublimation and

desorption

� 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

Spray Dryer

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.

� 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

Advantages

� 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.

Limitations:

� The equipment is very bulky, connected to accessories, fans,

heaters,)

� That it makes it expensive.

Applications:

� Drying in solution or in suspension form.

Limitations and 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.

� 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.

Freeze Drying

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 .

Freezing stageVacuum application

stagePrimary Drying

Freeze Drying Process - Stages

Secondary dryingPackaging

� 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.

Advantages

� 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.

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.

Limitations and Applications

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.

Product Solution

Primary Drying

Heat Input, Pressure

VFD PROCESS

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Dry Product

Secondary Drying

Heat Input, Pressure

Vacuum sealing

Heto Lyophilizer

� 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

Xerovac Drying

�

� 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.

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%)

Comparison of Xerovac and Freeze Drying :

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

� 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.

Super Critical Fluid Drying

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

Refrigeration:

� Freezing is best for long-term storage.

Low temperature:

� Reduces microbial growth and metabolism.

STORAGE

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� Reduces microbial growth and metabolism.

� Reduces thermal or spontaneous denaturation.

� Reduces adsorption on to the container wall.

� Smooth glass walls best to reduce adsorption or precipitation.

� Avoid polystyrene or containers with silanyl or plasticizer

coatings.

PACKAGINGPACKAGING

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coatings.

� Dark, opaque walls reduce chances of oxidation.

� Air-tight containers or argon atmosphere reduces air oxidation.

Vacuum Foam Drying (VFD)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� VFD commercialized in 90’s

1. Victor Bronshtein;

Universal Dying Technology, USA

2. Bruce Roser

Quadrant Healthcare, USA (Now VitriLife®)

Vacuum Foam Drying Steps

Bubble Formation Foam Formation

Foam Aging

Preconditioning

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 agentPluronics 0.1 – 3.0% Foaming agent

Inorganic water soluble

phosphate/s

0.5-5.0% Glass forming agent

Methyl paraben 0.01-0.15% Preservative

Sodium chloride As required Isotonicity adjusters

SWFI Q. S. Vehicle

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

Product Development Process (VFD)

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)

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

� 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.

Final Comments

� 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.