Development of Biopharmaceuticals and Biosimilar Drug Delivery

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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_at_yahoo.co.in
• Cell No 00919742431000

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Development of NDA and BLA
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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.

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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)

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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

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Biopharmaceuticals history
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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 gt 50 amino acids
• peptide lt 50 amino acids
• Function of a protein determined by its
  non-covalent 3D structure

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Covalently linked Amino Acids
Polypeptides
Amino Acids
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Peptide Synthesis
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Protein Structure
Lactate Dehydrogenase Mixed a / b
Immunoglobulin Fold b
Hemoglobin B Chain a
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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

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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

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Protein Pharmaceuticals

• Insulin (diabetes)
• Interferon b (relapsing MS)
• Interferon g (granulomatous)
• TPA (heart attack)

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Protein Pharmaceuticals

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

• Actimmune (If g)
• Activase (TPA)
• BeneFix (F IX)
• Betaseron (If b)
• Humulin
• Novolin
• Pegademase (AD)

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Protein Pharmaceuticals

• 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

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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

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Problem with Proteins (in vivo in the body)

• Elimination by B and T cells
• Proteolysis by endo/exo peptidases
• Small proteins (lt 30 kD) filtered out by the
  kidneys very quickly
• Unwanted allergic reactions may develop (even
  toxicity)
• Loss due to insolubility/adsorption

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Problem with Proteins (in vitro in the bottle)

• Noncovalent Covalent
• - Denaturation - Deamidation
• - Aggregation - Oxidation
• - Precipitation - Disulfide exchange
• - Adsorption - Proteolysis

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Noncovalent Processes

Denaturation
Adsorption
Aggregation
Precipitation
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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
  RS-SR RS- (Cys)
• Proteolysis - Asp-Pro, Trypsin (at Lys) or
  Chymotrypsin (at Phe/Tyr)

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How to Deal with These Problems

• Storage

Formulation
Delivery
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Storage

• Refrigeration
• Packaging
• Additives
• Freeze-Drying

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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

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Storage (Freeze Drying)

• Freeze liquid sample in container
• Place under strong vacuum
• Solvent sublimates leaving only solid or
  nonvolatile compounds
• Reduces moisture content to lt0.1

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How to Deal with These Problems
Storage

• Formulation

Delivery
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Protein Formulation

• Protein sequence modification (site directed
  mutagenisis)
• PEGylation
• Proteinylation
• Peptide Micelles
• Formulating with permeabilizers

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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.)

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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

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Peptide-PEG monomers
Hydrophobic block
Hydrophobic block
Peptide
Peptide
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Proteinylation

Protein Drug ScFv (antibody)
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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

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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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How to Deal with These Problems
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

• 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

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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

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Drug Delivery
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Novel Drug Delivery

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

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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

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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

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Polymeric Drug Delivery

• Controlled Release of drugs

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Polymeric Drug Delivery

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

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Polymeric Drug Delivery

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

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Polymeric Drug Delivery

• Drug Release by Polymer Degradation
• Polymer degradation by
• Hydrolysis
• Enzymatic (Phosphotases Proteases etc.)

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Microsphere Encapsulation
100 mm
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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

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Types of Microspheres

• Nonbiodegradable
• ceramic particles
• polyethylene co-vinyl acetate
• polymethacrylic acid/PEG
• Biodegradable (preferred)
• gelatin
• polylactic-co-glycolic acid (PLGA)

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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

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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)

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Release Mechanisms
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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

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Magnetic Targeted Carriers (MTCs)
Source http//www.magneticsmagazine.com/e-prints/
FeRx.htm
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Magnetic Targeted Carriers (MTCs)

• 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

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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

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Hydrogel Based Drug Delivery

• Hydrogels are three dimensional networks of
  hydrophilic
• polymers that are insoluble

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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

Insulin has been one drug that has been
incorporated in hydrogels and investigated
by researchers extensively
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Proteins in Pumps
Infusaid Model 400 Implantable Pump
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Proteins in Pumps
Mechanical Insulin Pumps
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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

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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

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Oral Delivery by Microsphere
pH 2 pH 7
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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)

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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

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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

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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

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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

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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

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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)

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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

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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

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Protein X

• Natural protein
• Specific enzymatic activity
• Negligible side effects
• Frequent injections (up to twice a day)
• Expensive

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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 deliveryimproved patient compliance
• May improve protein X utilization
• Stability of protein X in polymer?

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Protein X delivery

• Economical advantages
• Improved protein utilization
• Less protein gets wasted
• Drives down product cost
• Improved patient compliance
• Reduced frequency of dosing
• Improved patient compliance
• Less medical expenditure from
• events due to missed doses

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• BIOSIMILARS

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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

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What is a biosimilar 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

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What is a biosimilar 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
  physico-
• chemical methods
• Immunogenicity
• Structural heterogeneity
• Relatively high biological activity
• Relatively unstable

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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

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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

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Main elements CHMP guidelines concerning
biosimilars

• 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

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Main elements CHMP guidelines concerning
biosimilars

• 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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Thank You