Development of Biopharmaceuticals and Biosimilar Drug Delivery Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D KLE University’s College of Pharmacy Belgaum-590010

Development of Biopharmaceuticals and Biosimilar

Drug Delivery

Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D

KLE University’s College of Pharmacy Belgaum-590010E-mail: [email protected]

Cell No: 00919742431000

Development of NDA and BLA

24 Jan. 2010 Modern College of Pharmacy, Pune 2

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.

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, Parkinson’s 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


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 structure24 Jan. 2010 7Modern College of Pharmacy, Pune

Covalently linked Amino Acids

H3N+

O

O

RH

O

R3HNH

O

R4HH3N+

O

R1HNH

O

R2HNH

O

Amino Acids

Polypeptides

24 Jan. 2010 8Modern College of Pharmacy, Pune

Peptide Synthesis


Protein Structure

Lactate Dehydrogenase: Mixed /

Immunoglobulin Fold:

Hemoglobin B Chain:


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 (relapsing MS)

• Interferon (granulomatous)

• TPA (heart attack)24 Jan. 2010 13Modern College of Pharmacy, Pune


• Actimmune (If g)

• Activase (TPA)

• BeneFix (F IX)

• Betaseron (If b)

• Humulin

• Novolin

• Pegademase (AD)

• Epogen

• Regranex (PDGF)

• Novoseven (F VIIa)

• Intron-A

• Neupogen

• Pulmozyme

• Infergen24 Jan. 2010 14Modern College of Pharmacy, Pune


• 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/yr24 Jan. 2010 15Modern College of Pharmacy, Pune

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 quantities24 Jan. 2010 16Modern College of Pharmacy, Pune

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/adsorption24 Jan. 2010 17Modern College of Pharmacy, Pune


Problem with Proteins (in vitro – in the bottle)

Noncovalent Covalent

- Denaturation - Deamidation

- Aggregation - Oxidation

- Precipitation - Disulfide exchange

- Adsorption - Proteolysis


Noncovalent Processes

Denaturation Adsorption Aggregation Precipitation


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- + R’S-SR’’ goes to RS-SR’’ + R’S- (Cys)

• Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr)24 Jan. 2010 21Modern College of Pharmacy, Pune

How to Deal with These Problems

Storage

Delivery

Formulation


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

Delivery

Formulation


Protein Formulation

• Protein sequence modification (site directed

mutagenisis)

• PEGylation

• Proteinylation

• Peptide Micelles

• Formulating with permeabilizers24 Jan. 2010 27Modern College of Pharmacy, Pune

Site Directed Mutagenesis

E343H


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

| | | | | | | | | |O

H O

H O

H O

H O

H O

H O

H O

H O

H O

H


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 sites24 Jan. 2010 31Modern College of Pharmacy, Pune

Peptide-PEG monomers

Peptide Peptide

O

R3HNH

O

R4HH3N+

O

R1HNH

O

R2HNH

O

O

R3HNH

O

R4HH3N+

O

R1HNH

O

R2HNH

O

Hydrophobic block Hydrophobic block


Proteinylation

+

Protein Drug ScFv (antibody)


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


Peptide Micelles


Targeted Micelles


Formulation with permeabilizers

• Salicylates (aspirin)

• Fatty acids

• Metal chelators (EDTA)

• Anything that is known to “punch holes” into the intestine or lumen



Storage

Delivery

Formulation


Drug Delivery

• Non-conventional way of administering drugs (novel drug delivery)

• Conventional way • Oral (Tablets, Capsules)

• Parenteral (IV injections)


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


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

D R U G D E L IV E R Y C L A S S IF IC A T IO N

P u lm o n a ry P a re nte ra l T ra n sd e rm al

Im p la n ts O c u lar N a sal

M isce lla ne o us O ra l

R o u te o f A dm in is tra tion

P E G y la tion P ro -d ru g P o ly m er d e p ot

D ru g M o d ific a tion

D ru g D e liv e ry


Novel Drug Delivery

• Useful for following types of drugs:– Short half-life

• Insulin t1/2 < 25 min

• Growth hormone t1/2 < 25 min

– High systemic toxicity (causing side effects)• Carmustine causes nausea, hair loss

– Frequent dosing• Growth hormone Daily dosage required

– Expensive drugs24 Jan. 2010 45Modern College of Pharmacy, Pune

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

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MTC



• Polymers should be:– Biodegradable– Bio-compatible– Non-toxic

• Examples:– Polylactides/glycolides– Polyanhydrides– Polyphosphoesters



• Diffusion of drug out of the polymer• Governing equation: Fick’s laws of diffusion

• Drug release is concentration dependant

• Less applicable for large molecules

o o oo o oo o oo

o o oo o



• Drug Release by Polymer Degradation

• Polymer degradation by:• Hydrolysis

• Enzymatic (Phosphotases; Proteases etc.)


Microsphere Encapsulation

100 m24 Jan. 2010 52Modern College of Pharmacy, Pune

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)24 Jan. 2010 56Modern College of Pharmacy, Pune

Release Mechanisms

D iffu s io n

E n z y m a tic d e g rad a tion

B u lk e ro s ion S u rfa c e e ro s ion

H y d ro ly s i s C o m bin a tion

P o ly m e r D e g ra d a tion C o m bin a tion

D ru g R e le a se


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

Hydrophobic

Hydrophilic

Spherical vesicles with a phospholipid bilayer


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


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

R

O

N N

O

R

H H

R = polymer backbone

R

O

NH2+

O

HH

H2O

Insulin has been one drug that has been incorporated in hydrogels and investigated by researchers extensively


Proteins in Pumps

Infusaid Model 400 Implantable Pump


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


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 pH 724 Jan. 2010 70Modern College of Pharmacy, Pune

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 size24 Jan. 2010 72Modern College of Pharmacy, Pune

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


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)


Patch Delivery


Transdermal Patches

Micro fabricated needles to facilitates permeation of peptide drugs


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

• Improved patient compliance– Reduced frequency of dosing

– Improved patient compliance

– Less medical expenditure from

events due to missed doses0

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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


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

Factors influencing activity of therapeutic proteins


• Gene and promotor

• Host cell

• Culture conditions

• Purification

• Formulation

• Storage and handling

• Unknown factors

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


Pioneer Company Products Indication(s)US Patent/MarketExclusivityExpires

EU Patent/MarketExclusivityExpires

Genentech NutropinTM (somatropin) Growth disorders Expired ExpiredAbbott AbbokinaseTM (eudurase

urokinase)Ischemic events Expired Expired

Eli Lilly HumulinTM (recombinant insulin) Diabetes Expired ExpiredGenzyme Ceredase TM (algucerase):

Cerezyme TM (imiglucerase)Gaucher disease Expired Expired

AstraZeneca Streptase TM (streptokinase) Ischemic events Expired ExpiredBiogen/Roche Intron ATM (IFN-alfa-2b) Hepatitis B and C Expired ExpiredSerono Serotim TM (somatropin) AIDSwasting Expired NAEli Lilly Humatrope TM (somatropin) Growth disorders Expired

ODENA

Amgen EpogenTM, Procrit TM, EpresTM(erythropoietin)

Anemia 2013 Expired

Roche NeoRecormonTM (erythropoietin) Anemia NA ExpiredGenetech TNKaseTM (tenecteplase TNK-tPA) Acute myocardial infarction Expired ExpiredInterMune ActimmuneTM (IFN-gamma-Ib) Chronic granulomatous

Disease (CGD), malignant obsteopetrosis

ExpiredExpired2012

ExpiredExpired

Genentech Activase TM, Alteplase TM (tPA) Acute myocardial infaretion Expired2010

Expired

Chiron ProleukinTM (IL-2) HIV Expired, 2012

Expired

Amgen NeupogenTM (filgrastim G-CSF) Anemia, leukemia, neutropenia Expired, 2012, 2015

Expired

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

Thank You


Documents

Development of Biopharmaceuticals and Biosimilar Drug Delivery Dr. Basavaraj K. Nanjwade M.Pharm., Ph.D KLE University’s College of Pharmacy Belgaum-590010