Biotechnological product protein and peptide

Biotechnological Product
Protein and Peptide
Saroj V. Makwana

List of content:
 Introduction
 What is protein and peptide?
 Structure of protein and peptide
 Classes of therapeutic proteins and peptides:
 Problems with proteins
 Protein and peptide drug delivery
14-10-2017B.K.Mody Govt.pharmacy college,Rajkot
2

Introduction
 With the strong growth in biologics, large molecules, and biopharmaceutical
therapeutics in recent years, the pharmaceutical and biotech industries are
increasingly turning toward peptides and proteins in the search for drug discovery
targets.
 While both possess numerous properties that offer significant therapeutic potential,
there are fundamental differences between the two compounds.
3

What is Protein and Peptide?
 A Peptide contains less than 20 amino acids, having a molecular weight less than
5000, while a Protein possesses 50 or more amino acids and its molecular weight
lies above this value.
 Large biological molecules.
 Perform a vast array of functions within living organisms,including catalyzing
metabolic reactions, replicating DNA ,responding to stimuli, and transporting
molecules from one location to another.
4

Structure of Proteins and Peptides:
5

Mechanism of Protein transport:
6

Classes of Therapeutic Proteins and Peptides:
7
CLASS SUB CLASS ACTION/USE
Cardiovascular active peptides Angiotension II antagonist Treatment of heart failure in
patients intolerant of ACE
inhibitor therapy
Bradykinin Dilate blood vessel
CNS active peptides Delta sleep inducing factor Sleep regulation
Melanocyte-stimulating
hormones (MSH)
Class of peptide hormones that
are produced by cells in the
intermediate lobe of
the pituitary gland
Gastrointestinal active
peptides
Somatostatin Peptide hormone that regulates
the endocrine system
Gastrin antagonist Peptide hormone that
stimulates secretion of gastric
acid (HCl) by the parietal
cells of the stomach
Immunomodulating peptides Cyclosporine Prevent transplant rejection

8
Metabolism modulating
peptides
HGH Growth hormone
LHRH Trophic peptide hormone
responsible for the release of
follicle-stimulating hormone
(FSH) and luteinizing
hormone (LH) from the
anterior pituitary.
Monoclonal antibodies Omalizumab Antibody originally designed
to reduce sensitivity to
inhaled or ingested allergens,
especially in the control of
moderate to severe
allergic asthma

14-10-2017
9
9
These agents are
inactive orally
Some larger proteins
are taken by liver
Proteolytic cleavage
can inactivate
proteins/peptides.
Small proteins and all
peptides are rapidly
filtered by kidneys and
lost in urines
Insolubility leads
to aggregation
Intravenous injection
produce very high blood
level initially
The immune system
also clear foreign
proteins and may
cause severe reaction
Sub-cutaneous
injection may loss up
to 80% of the dose.
Problem with Proteins
(in vivo – in the body)

Problem with Proteins
(in vitro)
 Physical instability.
 Denaturation
 Adsorption
 Aggregation & Precipitation :
• Depends upon the relative hydrophilicity of surfaces
• air –water interphase
 Chemical instability.
 Oxidation & Reduction
 Proteolysis
 Di-sulphide exchange
 Racemization: Increase in susceptibility of peptide bonds toward proteolytic
enzymes.
10

11

Strategies for delivery Protein and Peptides
drugs:
 Modifying the physicochemical nature of macromolecule.eg prodrugs and analogs
of biologicals might protect them from degradation by proteases and other enzymes
present in the GI tract.
 Adding novel functionality to macromolecules For example, by attaching a drug
to a dipeptide that is recognized by a peptide-influx transporter, its oral
absorption can be increased. Efflux transporters such as P-glyco protein might
contribute significantly to the poor bioavailability of certain drugs, including
peptides.
 Using particulate delivery carrier systems So far, polymeric drug delivery
systems based on hydrogels, nanoparticles, microspheres, and lipid-based drug
delivery systems (e.g. microemulsions, liposomes, and solid lipid nanoparticles)
have been developed and employed for oral macromolecular drug delivery.
12

Selection of Delivery technology for Proteins &
Peptides:
 Optimum delivery of proteins & peptides to specific cells / diseased
sites (cancer cells).
 Reduction in potential side effects (especially immune response).
 Improvement upon the benefit / risk ratio.
 Delivery at a controlled fashion to the pharmacological receptor.
 Protection of intactness of protein & peptide from the body and vice
versa until they reaches to their site of action.
13

Nasal delivery:
 Local effects
 Low doses
 Overcomes hepatic first pass metabolism
 More complete absorption
 More rapid attainment of therapeutic blood levels,
 Quicker onset of pharmacological activity fewer side effects,
 High total blood flow per cm3,
 Porous endothelial membrane is easily accessible, and drug is delivered directly
to the brain along the olfactory nerves.
14

Factors influencing Nasal drug absorption
Physicochem
ical
properties of
drug
• Lipophilic-hydrophilic balance
• Enzymatic degradation in nasal cavity
• Molecular size. -difficulty of high molecular weight drugs (>1,000 Da)
Delivery
Effect
• Formulation (Concentration, pH): pH of the nasal formulation should be
adjusted to 4.5–6.5
• Drugs distribution and deposition: Depends on delivery device, mode of
administration, physicochemical properties of drug molecule.
Nasal Effect
• Mucociliary clearance
• Cold, rhinitis.
• Membrane permeability
15

Ideal drug candidate for nasal delivery
 Appropriate nasal absorption properties
 No nasal irritation from the drug
 A suitable clinical rationale for nasal dosage forms, e.g. rapid onset of action
 Low dose, Generally, below 25 mg per dose
 No toxic nasal metabolites
 No offensive odours/aroma associated with drug
 Suitable stability characteristics.
16

Advantage
 Non-invasive
 Rapid
 Comfortable
 Bypasses the BBB and targets the CNS, reducing systemic exposure and thus
systemic side effects.
 Facilitates the treatment of many neurologic and psychiatric disorders.
 Works for a wide range of drugs.
 Rich vasculature and highly permeable structure of the nasal mucosa greatly
enhance drug absorption.
17

Advantage
 Problem of degradation of peptide drugs is minimized up to a certain extent.
 Easy accessibility to blood capillaries.
 Direct delivery of vaccine to lymphatic tissue and induction of a secretory
immune response at distant mucosal site.
 A porous endothelial basement membrane that poses no restriction to
transporting the drug into general circulation.
 Realization of pulsatile delivery of some drugs like human growth hormone,
insulin, etc., is higher with nasal drug delivery.
18

Limitations
 Concentration achievable in different regions of the brain and spinal cord varies
with each agent.
 Delivery is expected to decrease with increasing molecular weight of drug.
 Some therapeutic agents may be susceptible to partial degradation in the nasal
mucosa or may cause irritation to the mucosa.
 Nasal congestion due to cold or allergies may interfere with this method of
delivery.
19

Formulation approach
20
Bio adhesives
Powders
Sprays /
Drops

Spray
 Simplest and most convenient
 Must be given in a volume of 25–200 µL
 For example, the bioavailability of nasally administered desmopressin has been
significantly increased by sprays, compared with drops.
 Unit dose containers,
 Metered dose sprays,
 Compressed air nebulizers
21
Various metered nasal spray pump.

Current peptide and protein therapeutics now
amenable to intranasal delivery.
Peptide or protein Clinical indication
Arginine vasopressin Primary nocturnal enuresis
Bilivarudin Anticoagulant
Calcitonin Prostate cancer and endometriosis
Cetrorelix Osteoporosis
Enfuvirtide Antiviral HIV infusion inhibitor
Insulin Diabetes
FSH Fertility
22

23

Oral delivery:
 Oral administration of protein drugs, however, is extremely difficult
due to their extremely low bioavailability.
 Development of oral protein formulations requires overcoming
obstacles, such as low permeability of large molecules, lack of
lipophilicity, and inactivation or rapid enzymatic degradation in the
gastrointestinal (GI) tract.
24

25 Approaches used in oral
protein formulations
Absorption
enhancer
Enzyme
inhibitors
Mucoadhesive
polymers

Absorption enhancers
 Absorption enhancers are used to improve the permeation of the protein/peptide
drugs through the intestinal wall, absorption enhancers are used as a
formulation components.
 Various absorption enhancers includes detergents or surfactants, bile salts and
ca2+ chelating agents.
 E.g. fatty acids, salicylates, chelators.
 Co-administration of proteins with carrier molecules can enhance
bioavailability of proteins.
 For example, lipophilic carrier enhancers facilitated the absorption of proteins,
such as insulin, human growth hormone, calcitonin, and recombinant
parathyroid hormone (rPTH).
26

Enzyme inhibitors
 Various enzyme inhibitors are used to minimize the degradation of protein by
various proteolytic enzymes.
 To minimize degradation of proteins by various proteolytic enzymes, researchers
have used trypsin or chymotrypsin inhibitors, such as pancreatic inhibitor,
soybean trypsin inhibitor, camostatmesylate, and aprotinin.
 A new class of enzyme inhibitors, chicken and duck ovomucoids have been
recently identified.
 E.g. sodium glycocholate, bacitracin.
27

Mucoadhesive polymeric systems
 Could extend the residence time at the site of drug absorption.
 Maintain intimate contacts with the mucus to increase the drug concentration
gradient and ensure immediate absorption without dilution or degradation in the
luminal fluid.
 E.g. Polymeric micro particles loaded with insulin showed a rapid burst
release with high insulin absorption in the intestine, resulting in a greater
hypoglycaemic effect without detectable mucosal damage
28

29

Particulate carrier delivery systems:
,
,
30
Nanopartic
les
Microspher
es
LiposomesEmulsions

Emulsion
 Protect proteins from chemical and enzymatic breakdown in the intestinal
lumen.
 The solid-in-oil-in-water (S/O/W) emulsion developed by using lipophilic
surfactant-coated insulin avoided degradation and enhanced permeation through
the intestinal mucosa.
 This system showed hypoglycaemic activity over several hours after oral
administration to diabetic rate.
 Drawbacks: Physical–chemical instability in long-term storage so storage at low
temperatures.
 Drawbacks can overcome by developing the dry emulsion formulations prepared
by spray drying, lyophilization, or evaporation.
31

Liposomes
 Liposomes used to improve the absorption of proteins from the intestinal tract.
 E.g. liposomal system including both insulin and sodium taurocholate markedly
decreased blood glucose levels after oral administration, and showed a high in-
vitro/ in-vivo correlation in the Caco-2 cell monolayer model.
32

Microsphere
 Manufactured by phase separation co-acervation methods
 Poly (methacrylic acid-g-ethylene glycol) P(MAA-g-EG) microspheres
containing insulin were not swollen in the acidic condition of the stomach due
to the formation of intermolecular polymer complexes, and thus, insulin
within the microspheres could be protected from acidic and proteolytic
degradation.
 After the exposure to the neutral and basic pH environments in the small
intestine, dissociation of the complexes causes swelling of microspheres,
resulting in insulin release.
 Within 2 h after oral administration of insulin-containing microspheres,
strong dose-dependent hypoglycemic effects were observed in both normal
and diabetic rats.
 These pH-sensitive microspheres restrict the release of proteins to favorable
area of the GI tract.
33

Protein oral delivery technologies under
development by companies.
Company Systems Product Outcomes for Products currently
Name Absorption available or under
Development
Emisphere Carrier molecules Eligen® Increase membrane Calcitonin, GPL-1,
permeability PYY, Insulin, Growth
hormone, parathyroid
hormone, heparin
Altus Protein CLEC® Stable against Calcitonin, other
crystallization proteolysis and self- polypeptides, Lipases,
Digestion esterases, and
Proteases
BioSante Calcium BioOral™ Protect proteins from Insulin and vaccines
Phosphate acidic degradation and
Nanoparticles improve membrane
permeability
Generex Spray device and Oral-Lyn™ Penetrate the buccal Insulin, Macrotonin
aerosol particles Epithelium
NOBEX/ Amphiphilic HIM2 Resist enzyme Insulin, enkephalin,
Biocon Oligomers digestion and increase calcitonin, parathyroid
membrane permeation Hormone
Apollo Nanoparticles Oradel™ Protect proteins from Insulin and TNF
Life enzyme digestion in blocker
Science the stomach and
facilitate the transport
of proteins in the
Intestine
Eli-Lilly Oral formulation AI-401 Protect proteins from Insulin
enzyme digestion
Provalis Lipid-based Macrulin™ Protect proteins from Insulin, salmon
PLC microemulsion proteolysis or acidic calcitonin
degradation, and
enhance the protein
absorption in GIT
Endorex Polymerizedlipos Orasome™ Protect proteins from Insulin and growth
Omes the stomach and upper hormone, vaccines
GIT
34

35

Pulmonary delivery
 Lungs possess a large surface area
 Extensive vascular network that favors absorption.
 Inhalation delivery is non-invasive.
 Avoids the first pass hepatic metabolism that orally delivered drugs
 To deposit successfully in the airways and alveoli, the aerodynamic diameter of
particles and droplets should be < 5 mm.
36

AERx® Pulmonary Delivery Technology
 Converts aqueous solutions of drugs or biologics into fine respirable
aerosols in 1–2 seconds in a highly efficient and reproducible manner
 Novo Nordisk, is currently in phase III development of AERx Insulin
Diabetes Management System® (AERx iDMS) which delivers insulin to
the systemic circulation via the lung.
37

Examples of Proteins/Peptides for Inhalation
Disease State Peptide/Protein
Local
Adult Respiratory Distress Surfactant Proteins (approved)
Syndrome
Cystic fibrosis (CF) DNase (approved)
Emphysema/CF Alpha-1-antitrypsin
Secretory leukoprotease inhibitor
Lung transplant Cyclosporin A
Cancer/Pneumocystis carnii Interferon-γ, Interleukin-2
38

Systemic
Osteoporosis Calcitonin Parathyroid hormone
Growth deficiency Human growth hormone
Multiple sclerosis Interferon-β
Diabetes Insulin
Cancer LH-RH analogs
Viral infections Ribavirin, Interferon-α
Neutropenia rhG-CSF
Anemia Erythropoetin
Anticoagulation Heparin
Diabetes insipidus dDAVP (1-deaminocysteine-8-
D-arginine vasopressin)
39

Colonic delivery:
 Greater responsiveness to absorption enhancers, protease inhibitors, and
novel bio adhesive and biodegradable polymers.
 Colon have advantage:- Having low almost nil peptidase activities
 Disadvantages:-Less surface area for absorption than small intestine.
 High concentration of anaerobic bacteria in the colon leads to faster
degradation of majority of drugs.
 Degradation by colonic microflora had been explored by the scientists to
reach site-specific delivery of peptide and proteins
40

 For the approach, the mechanisms like coating of molecules with azo-
aromatic groups or conjugation of molecule with azo-aromatic groups has
been implemented in which the entire molecule becomes impermeable to
the upper GIT and reaches the colon where through the action of microbial
flora, the molecule is released.
 This polymeric system was demonstrated to protect and deliver orally
administered insulin and vasopressin in rats.
41

42

Transdermal protein drug delivery:
 Transdermal delivery offers a potential alternative, overcoming set backs
associated with oral as well as injectable formulations.
 The passive delivery of proteins through the skin has to date been
impracticable due to their macromolecular and hydrophilic nature, which
limits their transport through molecules through skin.
 Amongst these, the use of chemical enhancers, iontophoresis,
phonophoresis and microporation have shown considerable promise.
43

Challenges
Penetration Barrier
 Barrier function of the skin is
achieved by the stratum corneum.
Due to its lipid content, the
permeability of the stratum
corneum to hydrophilic
molecules is limited, so highly
hydrophilic molecules, like
proteins and peptides, are unable
to partition into the lipoidal
stratum corneum layers and hence
cannot passively permeate
through skin.
 The diffusivity of a molecule
decreases exponentially with its
molecular volume and hence its
molecular weight.
Enzymatic Barrier
 Delivery of peptides and proteins is
mainly limited by proteolytic
enzymes. The epidermal and dermal
layers of the skin contain a variety of
endopeptidases, which cleave peptide
bonds within the peptide structure,
and exopeptidases which cleave the
N- or C-terminal peptide bonds.
 Endopeptidases -collagenases,
elastases, fibrinolysins, casenolytic
enzymes and enzymes of the
kallikreinekinin system
44

Techniques enabling transdermal delivery of
peptides and proteins
 Formulation Approaches:
45
• Encapsulation of a protein or peptide into a colloidal carrier is an
attractive approach for enhancing its transdermal delivery.
• Carrier systems such as conventional and specialized types of
liposomes and different types of nanoparticles are being widely
investigated to assist peptide delivery into and through the skin.
• e.g.transdermal delivery of insulin.
Encapsulation
Technologies
• Proteins and peptides are hydrophilic molecules and hence not
inherently suitable for transdermal delivery
• lipophilic prodrugs can be made by conjugating proteins with
lipophilic moieties.
Prodrugs
• Help in bypassing the enzymatic barrier of the skin
• Enzyme activity is inhibited by tight binding or covalent linkage of
the inhibitor to active sites on the enzyme.
• Inhibitors may also act by chelation of metal ions essential for
activity of the proteolytic enzyme.
Protease
Inhibitors

Chemical Permeation Enhancers
 Alcohols, polyalcohols, esters, fatty acids, pyrrolidones, sulfoxides, amines,
amides, surfactants and phospholipids.
 The mechanism of permeation enhancement differs for different molecules.
 For example, fatty acids act by fluidizing stratum corneum lipid bilayers,
while alcohols act by extracting stratum corneum lipids.
 Polar co-solvents such as propylene glycol help in solubilizing the
enhancers in the stratum corneum layers and have been shown to have a
synergistic action with enhancers like fatty acids.
46

Microporation:
 Microporation refers to the creation of micron sized channels through the
skin to assist transdermal delivery of molecules.
 These channels bypass the stratum corneum barrier and reach into the
epidermis layer. However, they do not reach the dermis where nerves and
blood vessels are located.
 The microchannels are typically large in dimension compared to any drug
molecule. Since macromolecules like proteins have dimensions in the nano
range, they can be easily delivered through these microchannels into and
across skin.
 Painless, non-invasive and yet an effective and elegant method to achieve
enhanced delivery of molecules through the skin.
47

• Several academic and industrial groups are actively involved in the
development of mechanical microneedles, including 3M, Corium, Becton
Dickinson, the Georgia Institute of Technology and Zosano Pharma.
• Microneedles have been created from a variety of materials including
silicon, metals, glass and polymers, as well as from sugars such as maltose
which can dissolve when inserted in skin.
• Microneedles of dimensions varying from 300-900 mm have also been
fabricated from commercially available 30G hypodermic needles, by a
method where the needles were placed at predetermined lengths through
holes created in a polyetheretherketone mold and then cut and glued at the
back of the mold.
48

49
A
B
C
D
Microneedle array assembled from commercially available 30G hypodermic needles. (A)
Tip of a 30G hypodermic needle; (B) The assembled microneedle array; (C) Microneedle
array applicator in comparison with a ruler; (D) Applicator at an angled view.

Iontophoresis:
 Non-invasive, electrically assisted technique in which a physiologically acceptable
amount of electric current (up to 0.5 mA/cm2) is used to facilitate transdermal
delivery of charged and neutral molecules.
 Unlike other physical enhancement techniques, which tend to disrupt the skin
barrier in promoting transdermal flux, iontophoresis acts on the drug molecule
itself.
 These molecules are propelled into the deeper layers of the skin under the influence
of an electric current.
 Depending on the physicochemical nature of the drug, mechanisms involving
electrorepulsion or electro-osmosis predominate to drive the molecule into and
across skin.
 Electrorepulsion facilitates the delivery of charged molecules; when they are placed
under a similarly charged electrode, repulsion between the like charges of the
electrode and the therapeutic molecule tend to drive the molecule through the skin.
50

51
A schematic representing iontophoresis carried out on skin. In this figure, a positively
charged drug molecule is placed under the positively charged electrode (anode).
Repulsion between like charges drives the molecule through the skin (bracketed).

Electroporation
 Electroporation involves exposing the skin to very high voltages (w10e1000 V) for
very short durations of time (1-100 ms).
 Used for introducing DNA material into cells, before its use in transdermal
applications.
 It causes transient structural changes to the upper skin layers, thereby creating
aqueous pathways across the skin.
 Enhancement in the permeation of molecules into and across skin occurs by several
mechanisms, such as improved diffusion, electrophoretic movement and electro-
osmosis.
 This technique has been effective in assisting and enhancing transdermal delivery of
peptides and proteins.
 Application of electroporation prior to iontophoresis was shown to enhance the flux
of luteinizing hormone releasing hormone (LHRH) across the isolated perfused
porcine skin flap model. LHRH delivery increased with an increase in the number
of electroporative pulses.
52

Published reports on peptide transdermal
delivery
Molecular
Peptide name Skin type Weight(gm/mol) References
A.J. Hoogstraate et
Leuprolide In-vitro, human skin 1209 al.
Buserelin In-vitro, human skin 1239 E. Knoblauch et al.
vasopressin In-vitro, human skin - J.W. Sharkey et al.
In-vitro, hairless mouse
Desmopressin skin 1069 M. Martin et al.
Angiotensin 2 In vitro, human skin 1046 M. Clemmesy et al.
In-vitro, hairless mouse
Calcitonins skin - B. Kari et al.
Octreotide In-vivo, rabbits 1019 J.W. Sharkey et al.
Salmon
calcitonin In-vivo, rats 3509 K. Morimoto et al.
Human calcitonin In-vivo, hairless rats 3527 S. Thysman et al.
53

54

Ocular drug delivery
 Conventional drug delivery systems such as solutions, suspensions,
gels, ointments and inserts have been investigated for controlled
ocular delivery, they suffer from problems such as poor drainage of
instilled solutions, tear turnover, poor corneal permeability,
nasolacrimal drainage, systemic absorption and blurred vision.
 Advanced drug delivery systems have been developed with the
intention of optimizing and controlling delivery of ocular
therapeutics to the target sites, either by increasing its penetration
across the mucosa or by prolonging the contact time of the carrier
with the ocular surface, and have shown promising results.
55

Barriers in ocular delivery of proteins
56

Examples of therapeutic peptides delivered
through ocular route.
Disorder/Indication Therapeutic peptide
Antiallergic, anti-inflammatory ACTH
Analgesic Endorphin, Leu-enkephalin
Antiscarring agent in glaucoma Integrin-binding peptide
filtration surgery
Attenuate miotic response Somatostatin
Choroidal or retinal Octreotide, Urokinase derived peptide,
neovascularization Cyclic integrin-binding peptide
Corneal epithelial wound Insulin-like growth factor derived peptide
Substance P derived peptide
Diabetes mellitus Insulin
Diabetes insipidus Vasopressin
Diagnosis of thyroid cancer TSH
Dry eye disease Cyclosporine A
57

References
 Aradigm Corporation, Protein Delivery – Consider the Pulmonary Route:
touchbriefings 2007.gy & S
 Sally-Ann Cryan. Carrier-based Strategies for Targeting Protein and Peptide Drugs to
the Lungs: The AAPS Journal 2005; 7 (1) Article 4 (http://www.aapsj.org).
 Christopher Cullander and Richard H. Guy. Transdermal delivery of peptides and
proteins: Advanced Drug Delivery Reviews, 8 (1992) 291 329
 M. Foldvari, M.E. Baca-Estrada, Z. He, J. Hu, S. Attah-Poku, M. King, Dermal and
transdermal delivery of protein pharmaceuticals: Lipid-based delivery systems for
interferon alpha, Biotechnol. Appl. Biochem. 30 (1999), 129-137.
 Kyeongsoon Park, Ick Chan Kwon, Kinam Park. Oral protein delivery: Current status
and future prospect: Reactive & Functional Polymers 71 (2011) 280–287.
 Sy-Juen Wu, Joseph R. Robinson. Transport of human growth hormone across Caco-2
cells with novel delivery agents: evidence for P-glycoprotein involvement: Journal of
Controlled Release 62 (1999) 171–177
58

 M.K. Marschutz, A. Bernkop-Schnurch, Biomaterials 21 (2000) 1499–1507.
 Reza Aboofazeli, Peptide and Protein Delivery at a Glance: Iranian Journal of
Pharmaceutical Research (2003) 1-2
 Arvind Sharma, Sandeep Arora, Commercial challenges and emerging trends in oral
delivery of peptide and protein drugs: A review: Research Journal of Pharmaceutical,
Biological and Chemical Sciences. July – September 2011, Volume 2 Issue 3 Page No.
778
 Hussain A, Hamadi S, Kagoshima M, Iseki K, Dittert L. Does increasing the
lipophilicity of peptides enhance their nasal absorption.J Pharm Sci 1991; 80: 1180-1181
 Satish BB, adhikrao VY, Amelia MA, Rajkumar M, Bio availability of intranasal drug
delivery system, Asian J of Pharmaceutics, 2008; 201-15.
 Cho JH, Kwun YS, Jang HS, Kang JM, Won YS, Yoon HR. Longterm use of
preservatives on rat nasal respiratorymucosa: effects of benzalkoniumchloride and
potassium sorbate. Laryngoscope 2000; 110: 312.7.
 Edward T Maggio, intravailTM highly effective intranasal delivery of peptide and
protein drug. Expert Opin.Drug Deliv. (2006) 3(4):529-539.
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