Developmentof Biopharmaceuticalsand Biosimilars Drug Delivery

Development of Biopharmaceuticals and Biosimilar Drug Delivery
Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D KLE Universitys College of Pharmacy Belgaum-590010
E-mail: bknanjwade@yahoo.co.in Cell No: 00919742431000
Development of NDA and BLA
24 Jan. 2010
Modern College of Pharmacy, Pune
What are Biopharmaceuticals

Biopharmaceuticals are defined as pharmaceuticals manufactured by biotechnology methods, with the products having biological sources, usually involving live organisms or their active components Biopharmaceuticals are protein or nucleic acid based pharmaceuticals (substance used for therapeutic or in vivo diagnostic purpose), which are produced by mean other than direct extraction from a native biological source.
24 Jan. 2010 Modern College of Pharmacy, Pune 3
Pharmaceutical Biotechnology
The methods and techniques that involve the use of living organisms (such as cells, bacteria, yeast and others) are tools to perform specific industrial or manufacturing process are called biotechnology Pharmaceutical Biotechnology will continue to provide new breakthroughs in medical research in the years to come, leading to treatment in field which have previously eluded us (including AIDS, cancer asthma, Parkinsons disease, Alzheimer disease)
Pharmaceutical Biotechnology
Biotechnology offers better product-targeting for specific diseases and patient groups, through the use of innovative technologies, in particular, genetics. Examples include, amongst others, treatment for rare diseases and cancers. Some products are not naturally created in sufficient quantities for therapeutics purpose. Biotechnology makes large-scale production of existing substances possible, for example, insulin in the field of diabetes treatment
Biopharmaceuticals history
24 Jan. 2010
Protein and peptide

Proteins - Chains of amino acids, each joined together by a specific type of covalent bond Proteins formed by joining same 20 amino acids in many different combinations and sequences Protein > 50 amino acids peptide < 50 amino acids Function of a protein determined by its non-covalent 3D structure
Covalently linked Amino Acids

O H H 3N+ R2 H N O H O H N H R4 O O
O H 3N+ H R Amino Acids

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R1
N H
R3
Polypeptides
Peptide Synthesis
24 Jan. 2010
Protein Structure
Lactate Dehydrogenase: Mixed E / F

24 Jan. 2010
Immunoglobulin Fold: F
Hemoglobin B Chain: E
10
Classification of Proteins
According to their biological roles - Enzymes Catalyses virtually all chemical reactions i.e. 6GDH
- Transport proteins i.e. Haemoglobin of erythrocytes - Contractile or Motile proteins i.e. Actin and Myosin - Structural proteins i.e.Collagen - Defense proteins i.e. Immunoglobulins and Antibodies - Regulatory proteins i.e. insulin - Nutrient and storage proteins i.e. Ovalbumin
Protein Therapeutics
Proteins/peptides are gaining prominence Proteins - ideal drugs as they carry out essentially all biologic processes and reactions Recombinant DNA, hybridoma techniques, scale fermentation and purification processes brought new series of Proteins/peptides
Protein Pharmaceuticals
Insulin (diabetes) Interferon F(relapsing MS) Interferon K (granulomatous) TPA (heart attack)
Actimmune (If g) Activase (TPA) BeneFix (F IX) Betaseron (If b) Humulin Novolin Pegademase (AD)
24 Jan. 2010
Epogen Regranex (PDGF) Novoseven (F VIIa) Intron-A Neupogen Pulmozyme Infergen

14
77 FDA approved protein drugs 66/77 are recombinant proteins Protein pharmaceutical sales currently approach $25 billion/yr By 2012 they are expected to reach $60 billion/yr
Challenges with Proteins

Very large and unstable molecules Structure is held together by weak non-covalent forces Easily destroyed by relatively mild storage conditions Easily destroyed/eliminated by the body Hard to obtain in large quantities
Problem with Proteins (in vivo in the body)

Elimination by B and T cells Proteolysis by endo/exo peptidases Small proteins (< 30 kD) filtered out by the kidneys very quickly Unwanted allergic reactions may develop (even toxicity) Loss due to insolubility/adsorption
24 Jan. 2010
18
Problem with Proteins (in vitro in the bottle)

Noncovalent
- Denaturation - Aggregation - Precipitation - Adsorption
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Covalent
- Deamidation - Oxidation - Disulfide exchange - Proteolysis
19
Noncovalent Processes
Denaturation
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Adsorption
Aggregation Precipitation
20
Covalent processes
Deamidation - conversion of Asn-Gly sequences to a-Asp-Gly or b-Asp-Gly Oxidation - conversion RSR to RSOR, RSO2R or RSO3R (Met & Cys) Disulfide exchange - RS- + RS-SR goes to RSSR + RS- (Cys) Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr)
How to Deal with These Problems
Storage
Formulation
Delivery
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22
Storage
Refrigeration Packaging Additives Freeze-Drying
Storage (additives)
Addition of stabilizing salts or ions (Zn+ for insulin) Addition of polyols (glycerol and/or polyethylene glycol) to solubilize Addition of sugars or dextran to displace water or reduce microbe growth Use of surfactants (CHAPS) to reduce adsorption and aggregation
Storage (Freeze Drying)

Freeze liquid sample in container Place under strong vacuum Solvent sublimates leaving only solid or nonvolatile compounds Reduces moisture content to <0.1%
Storage
Formulation
Delivery
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Protein Formulation
Protein sequence modification (site directed mutagenisis) PEGylation Proteinylation Peptide Micelles Formulating with permeabilizers
Site Directed Mutagenesis
E343H
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Site Directed Mutagenesis

Allows amino acid substitutions at specific sites in a protein i.e. substituting a Met to a Leu will reduce likelihood of oxidation Strategic placement of cysteines to produce disulfides to increase Tm Protein engineering (size, shape, etc.)
PEGylation
CH-CH-CH-CH-CH-CH-CH-CH-CH-CH | | | | | | | | | | OH OH OH OH OH OH OH OH OH OH
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PEGylation
PEG is a non-toxic, hydrophilic, FDA approved, uncharged polymer Increases in vivo half life (4-400X) Decreases immunogenicity Increases protease resistance Increases solubility & stability Reduces depot loss at injection sites
Peptide-PEG monomers
Hydrophobic block
Peptide
Hydrophobic block
Peptide
O H H3N+ H R1 N H
R2 H N O H
O H R3 N H
R4
O H O H3N+ H R1 N H
R2 H N O H
O H R3 N H
R4 O O
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Proteinylation
Protein Drug
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ScFv (antibody)
Modern College of Pharmacy, Pune 33
Proteinylation
Attachment of additional or secondary (nonimmunogenic) proteins for in vivo protection Increases in vivo half life (10X) Cross-linking with Serum Albumin Cross-linking or connecting by protein engineering with antibody fragments
Peptide Micelles
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Peptide Micelles
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Targeted Micelles
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Formulation with permeabilizers

Salicylates (aspirin) Fatty acids Metal chelators (EDTA) Anything that is known to punch holes into the intestine or lumen
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Storage
Formulation
Delivery
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Drug Delivery
Non-conventional way of administering drugs (novel drug delivery) Conventional way
Oral (Tablets, Capsules) Parenteral (IV injections)
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Conventional
ORAL
Ease of administration Patient Compliance Exposure to extremely acidic pH Poor absorption of larger drugs Degradation by enzymes
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INTRAVENOUS
Fast action No absorption issues Lesser patient compliance Fast clearance of drugs
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Parenteral Delivery of Proteins

Intravenous Intramuscular Subcutaneous Intradermal
Parenteral Delivery of Proteins

Route of delivery for 95% of proteins Allows rapid and complete absorption Allows smaller dose size (less waste) Avoids first pass metabolism Avoids protein unfriendly zones
Problems with overdosing, necrosis Local tissue reactions/hypersensitivity Everyone hates getting a needle
Drug Delivery
DRUG DELIVERY CLASSIFICATION
Drug Delivery
Route of Administration
Drug Modification
Pulmonary
Parenteral
Transdermal Implants
Miscellaneous
Oral
PEGylation
Pro-drug
Polymer depot
Ocular
Nasal
44
24 Jan. 2010
Novel Drug Delivery

Useful for following types of drugs:
Short half-life
Insulin Growth hormone Carmustine t1/2 < 25 min t1/2 < 25 min causes nausea, hair loss
High systemic toxicity (causing side effects) Frequent dosing

Growth hormone Daily dosage required
Expensive drugs
Novel Drug Delivery

Adverse Drug Effects
15 % of hospital admissions 100,000 deaths $136 billion in health care costs
Less patient compliance

10 % hospital admissions
Novel Drug delivery sales

$14 billion in 1997 & $53 billion in 2002
Polymeric Drug Delivery

Frequency of doses reduced Drug utilized more effectively Drug stabilized inside the polymer matrix Reduced side effects
Possibility of dose-dumping De-activation of drug inside polymer


Controlled Release of drugs
60
Plasma concentration
50 40 Conventional 30 20 10 0 0 1 2 3 4 5 6 7 8 Controlled release MEC MTC
Time

Polymers should be:
Biodegradable Bio-compatible Non-toxic
Examples:
Polylactides/glycolides Polyanhydrides Polyphosphoesters

Diffusion of drug out of the polymer
Governing equation: Ficks laws of diffusion Drug release is concentration dependant
o o o o o o o o o o o o o o o
Less applicable for large molecules


Drug Release by Polymer Degradation
Polymer degradation by:

Hydrolysis Enzymatic (Phosphotases; Proteases etc.)
Microsphere Encapsulation
100 Qm
Encapsulation
Process involves encapsulating protein or peptide drugs in small porous particles for protection from insults and for sustained release Two types of microspheres
nonbiodegradable biodegradable
Types of Microspheres
Nonbiodegradable
ceramic particles polyethylene co-vinyl acetate polymethacrylic acid/PEG
Biodegradable (preferred)
gelatin polylactic-co-glycolic acid (PLGA)
Microsphere Release
Hydrophilic (i.e. gelatin)
best for burst release
Hydrophobic (i.e. PLGA)

good sustained release (esp. vaccines) tends to denature proteins
Hybrid (amphipathic)
good sustained release keeps proteins native/active
Polymer Scaffolds
Incorporate drug into polymeric matrix Protection of drug from enzymatic degradation particularly Applicable to peptide and protein drugs Release drug at known rate over prolonged duration Drug dispersed or dissolved in suitable polymer Release - diffusion of drug through polymer - diffusion through pores in polymer structure - therefore different release profiles result (dissolved or dispersed)
Release Mechanisms
Drug Release
Diffusion
Polymer Degradation
Combination
Enzymatic degradation
Hydrolysis
Combination
Bulk erosion
24 Jan. 2010
Surface erosion
57
Magnetic Targeted Carriers (MTCs)

Microparticles, composed of elemental iron and activated carbon Drug is adsorbed into the MTCs and transported The drug attaches to the carbon component The particles serve as delivery vehicles to the area of the tumor for site-specific targeting
Source: http://www.magneticsmagazine.com/e-prints/FeRx.htm

FeRx Inc. is the leader in the development in this innovative technology Founder of FeRx and pioneer of magnetic targeted drug delivery is Dr. Kenneth Widder Began with albumin microspheres containing encapsulated drugs, and lead to present MTC technology Present clinical trials by FeRx show that drug remains for 28-days with no redistribution from the targeted site
Liposomes
Spherical vesicles with a phospholipid bilayer
Hydrophilic
Hydrophobic
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Liposomes Drug Delivery

Potential of liposomes in drug delivery has now realized Bloemycin encapsulated in thermosensitive liposomes enhanced antitumor activity and reduced normal tissue toxicity S.C injection of negatively charged liposomes produced a prolonged hypoglycemic effect in diabetic dogs Liposomes have recently been used successfully as vehicles for vaccines
Hydrogel Based Drug Delivery

Hydrogels are three dimensional networks of hydrophilic polymers that are insoluble
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63
Hydrogel Based Drug Delivery

Hydrogels can swell as a result of changes in pH, Temp., ionic strength, solvent composition, pressure and the application of electric fields
O R N H N H R = polymer backbone O R O H2O R NH2
+
H
O H
Insulin has been one drug that has been incorporated in hydrogels and investigated by researchers extensively
Proteins in Pumps
Infusaid Model 400 Implantable Pump
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65
Proteins in Pumps
Mechanical Insulin Pumps

Proteins in Pumps
Formulation is the beginning of successful drug delivery Multiple potential interactions between the protein and the pump Control of the material interface is most important Device design and formulation need to work together and be regulated together
Oral Protein Delivery
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Oral Insulin
Buccal aerosol delivery system developed by Generex Insulin is absorbed through thin tissue layers in mouth and throat Insulin is formulated with a variety of additives and stabilizers to prevent denaturation on aerosolization and to stabilize aerosol particles
Oral Delivery by Microsphere
pH 2
24 Jan. 2010 Modern College of Pharmacy, Pune
pH 7
70
pH Sensitive Microspheres
Gel/Microsphere system with polymethacrylic acid + PEG In stomach (pH 2) pores in the polymer shrink and prevent protein release In neutral pH (found in small intestine) the pores swell and release protein Process of shrinking and swelling is called complexation (smart materials)
Nasal Delivery of Proteins

Extensive microcirculation network underneath the nasal mucosa Drug absorbed nasally can directly enter the systemic circulation before passing through the hepatic circulation The nasal administration of peptides has attracted much interest now a days due to
- Relatively rapid absorption of drug
- Little metabolic degradation - Relative ease of administration - Selective to peptide structure and size
Nasal Delivery of Proteins

Enhancement of nasal absorption of insulin using polyacrylic acid as a vehicle Enhancement in the nasal absorption of insulin entrapped in liposomes through the nasal mucosa of rabbits Administration of insulin (1 IU/ kg) via the nasal route caused a significant decrease in the plasma glucose level The nasal route appears to be a viable means of systemically delivering many small peptides
Pulmonary Delivery
Deep lung, an attractive site of protein delivery due to - Relatively large surface area (100m2)
- Rapid absorption of drug into the blood stream through the alveoli
Dura and Inhale developed dry powder delivery systems for proteins 40% of the insulin administered in an aerosol, to the trachea of anaesthetized rabbit was absorbed Albumin was largely absorbed within 48 hours of instillation into the lungs of guinea pigs and dogs
Rectal Delivery
The rectal delivery offers many advantages - Avoidance of drug dilution prior to reaching the systemic
circulation - Reduction in first-pass metabolism - Rapid systemic absorption - Safe and convenient especially in case of neonates and infants - Greater dose may be administered - Withdrawal of drug is possible in case of adverse effects
Administration of insulin using the rectal route shows systemic absorption

Occular Delivery
Gelfoam eye device enhances the absorption of sodium insulin with an absorption enhancer Many proteins and peptides that have been investigated for ocular delivery - Enkephalins
- Thyrotropin releasing hormones, - Leutanizing hormone-releasing hormone, - Glucagon and Insulin
All these peptides were absorbed into the blood stream to some extent
Patch Delivery
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Mucoadhesive Patch
Adheres to specific region of GI tract Ethylcellulose film protects drugs from proteolytic degradation Composed of 4 layers
Ethylcellulose backing Drug container (cellulose, citric acid) Mucoadhesive glue (polyacrylic acid/PEG) pH Surface layer (HP-55/Eudragit)
24 Jan. 2010
Patch Delivery
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Transdermal Patches
Micro fabricated needles to facilitates permeation of peptide drugs
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Transdermal Patches
Proteins imbedded in a simple matrix with appropriate additives Patch is coated with small needles that penetrate the dermal layer Proteins diffuse directly into the blood stream via capillaries Less painful form of parenteral drug delivery
Role of a Pharmaceutical Engineer

Modeling of drug delivery systems
Prediction of kinetics/thermodynamics
Novel polymer research

Temperature sensitive polymers; pH sensitive polymers
Development of new drug delivery techniques

Novel techniques for new therapies
Development of purification processes

Solvent Removal; Removal of impurities etc.
Process development
Design & Development of robust processes; GMP Validation
Scale-up of processes
Protein X
Natural protein Specific enzymatic activity Negligible side effects Frequent injections (up to twice a day) Expensive
Protein X delivery
Applicable alternative techniques
Pulmonary delivery
Non-invasive; Good patient compliance Poor efficiency; Requires patient training
PEGylation
Improved stability; reduced frequency of injections Protein X activity?
Polymeric delivery
Long-term delivery;improved patient compliance May improve protein X utilization Stability of protein X in polymer?
Protein X delivery
Economical advantages
Improved protein utilization
Less protein gets wasted Drives down product cost
60 50 40 Conventional 30 Controlled release 20 10 0 0 1 2 3 4 5 6 7 8 MEC
Time (days)
Improved patient compliance

60
Reduced frequency of dosing Improved patient compliance Less medical expenditure from events due to missed doses
50 40 30 20 10 0 0 1 2 3 4 5 6 7 8
Time (days)
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BIOSIMILARS
What is a biosimilar medicine

A biosimilar medicine is a medicine which is similar to a biological medicine that has already been authorized (the biological reference medicine) The active substance of a biosimilar medicine is similar to the one of the biological reference medicine

Biosimilar and biological reference medicines are used in general at the same dose to treat the same disease Since biosimilar and biological reference medicine are similar but not identical The name, appearance and packaging of a biosimilar medicine differ to those of the biological reference medicine

As biosimilars are not generics, the generic substitution rules should not apply to biosimilars
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Characteristics of therapeutic proteins

Size - 100 500 times larger than classic drugs - Can not be completely characterized by physicochemical methods Immunogenicity Structural heterogeneity Relatively high biological activity Relatively unstable
Factors influencing activity of therapeutic proteins

Gene and promotor Host cell Culture conditions Purification Formulation Storage and handling Unknown factors
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What is in a name
Biogenerics Second entry biologicals Subsequent entry biologicals Off-patent biotech products Multisource products Follow-up biologics Biosimilars Similar biological medicinal products
24 Jan. 2010
Pioneer Company
Products
Indication(s)
US Patent/ Market Exclusivity Expires
EU Patent/ Market Exclusivity Expires
Genentech Abbott Eli Lilly Genzyme AstraZeneca Biogen/Roche Serono Eli Lilly Amgen Roche Genetech InterMune
NutropinTM (somatropin) AbbokinaseTM (eudurase urokinase) HumulinTM (recombinant insulin) Ceredase TM (algucerase): Cerezyme TM (imiglucerase) Streptase TM (streptokinase) Intron ATM (IFN-alfa-2b) Serotim TM (somatropin) Humatrope TM (somatropin) EpogenTM, Procrit TM, EpresTM (erythropoietin) NeoRecormonTM (erythropoietin) TNKaseTM (tenecteplase TNK-tPA) ActimmuneTM (IFN-gamma-Ib)
Growth disorders Ischemic events Diabetes Gaucher disease Ischemic events Hepatitis B and C AIDSwasting Growth disorders Anemia Anemia Acute myocardial infarction Chronic granulomatous Disease (CGD), malignant obsteopetrosis Acute myocardial infaretion HIV
Expired Expired Expired Expired Expired Expired Expired Expired ODE 2013
Expired Expired Expired Expired Expired Expired NA NA Expired
Genentech Chiron Amgen
Activase TM, Alteplase TM (tPA) ProleukinTM (IL-2)
NeupogenTM (filgrastim G-CSF) Anemia, leukemia, neutropenia 24 Jan. 2010 Modern College of Pharmacy, Pune
NA Expired Expired Expired Expired Expired Expired Expired 2012 Expired Expired 2010 Expired, Expired 2012 Expired, Expired 2012, 2015 93
Main elements CHMP guidelines concerning biosimilars

The concept of similar biological products is applicable to any biological medicinal product. But it is more likely applied to highly purified products, which can be thoroughly characterized In order to support pharmacovigilance monitoring, the specific product given to the patient should be clearly identified

The active substance of the biosimilar product must be similar in molecular and biological terms to the active substance of the reference medicinal product e. IFN alpha 2a is not similar to IFN alpha 2b The same reference product throughout the comparability program The pharmaceutical form, dose and route of administration of the biosimilar and the reference product should be the same

If the reference product has more than one indication, the safety and eficacy for all indications have to be justified or demonstrated for each indication separately The clinical safety must be monitored on an ungoing basis after marketing approval The issue of immunogenicity should always be addressed, and its long-term monitoring is necessary
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Development of Biopharmaceuticals and Biosimilar Drug Delivery

Development of NDA and BLA

Modern College of Pharmacy, Pune

What are Biopharmaceuticals

Modern College of Pharmacy, Pune

Protein and peptide

Covalently linked Amino Acids

O H 3N+ H R Amino Acids

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Lactate Dehydrogenase: Mixed E / F

Epogen Regranex (PDGF) Novoseven (F VIIa) Intron-A Neupogen Pulmozyme Infergen

Modern College of Pharmacy, Pune

Challenges with Proteins

Problem with Proteins (in vivo in the body)

Modern College of Pharmacy, Pune

Problem with Proteins (in vitro in the bottle)

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

How to Deal with These Problems

Modern College of Pharmacy, Pune

Storage (Freeze Drying)

How to Deal with These Problems

Modern College of Pharmacy, Pune

Site Directed Mutagenesis

Modern College of Pharmacy, Pune

Site Directed Mutagenesis

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Formulation with permeabilizers

Modern College of Pharmacy, Pune

How to Deal with These Problems

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Modern College of Pharmacy, Pune

Parenteral Delivery of Proteins

Parenteral Delivery of Proteins

Modern College of Pharmacy, Pune

Novel Drug Delivery

High systemic toxicity (causing side effects) Frequent dosing

Novel Drug Delivery

Less patient compliance

Novel Drug delivery sales

Polymeric Drug Delivery

Possibility of dose-dumping De-activation of drug inside polymer

Polymeric Drug Delivery

50 40 Conventional 30 20 10 0 0 1 2 3 4 5 6 7 8 Controlled release MEC MTC

Polymeric Drug Delivery

Polymeric Drug Delivery

Less applicable for large molecules

Polymeric Drug Delivery

Polymer degradation by:

Hydrophobic (i.e. PLGA)

Modern College of Pharmacy, Pune

Magnetic Targeted Carriers (MTCs)

Magnetic Targeted Carriers (MTCs)

Magnetic Targeted Carriers (MTCs)

Modern College of Pharmacy, Pune

Liposomes Drug Delivery