Transcript (Bioteh. Products).ppt

King Saud University
College of Pharmacy
Departments of Pharmaceutics/
Pharmacognosy
Pharmaceutical Biotechnology
PHG 424
Mounir M. Salem, Ph.D.
[email protected]
©1999 Timothy G. Standish
Biotechnology Products
©1999 Timothy G. Standish
Microbiological Consideration
 Most proteins are administered parenterally and have to be
sterile.
 In general proteins are sensitive to heat and other regularly
used sterilization methods; they can’t withstand autoclaving, gas
sterilization, or sterilization by ionizing radiation. Consequently,
sterilization of the end product is not possible.
Therefore, protein pharmaceuticals have to be assembled under
aseptic conditions.
©1999 Timothy G. Standish
Microbiological Consideration…cont.
 Equipment and excipients are treated separately and autoclaved,
or sterilized by dry heat (>160 ºC), chemical treatment or
radiation to minimize bioburden.
 As a recombinant DNA products are grown in microorganisms,
these should be tested for viral contamination and appropriate
measures should be taken if viral contaminations occur.
 Excipients with a certain risk factor such as blood derived,
human serum albumin should be carefully tested before use and
their presence in the formulation processes should be minimized.
©1999 Timothy G. Standish
Microbiological Consideration…cont.
 Bioburden or microbial limit testing is performed on
pharmaceutical products and medical products as a quality control
measure. Products or components used in the pharmaceutical or
medical field require control of microbial levels during processing
and handling.
 Bioburden of raw material as well as finished pharmaceutical
products can help to determine whether the product complies with
the requirements of the BP, Eur. or USP.
 Bioburden is the number of microorganisms with which an object
is contaminated. This unit is measured in CFU per gram of product.
©1999 Timothy G. Standish
Excipients used in biotechnology products
Introduction:
• Active ingredient.
• Solubility enhancers.
• Anti-adsorption and anti-aggregation agents.
• Buffer components.
• Preservatives and anti-oxidants.
• Osmotic agents.
• Carrier system.
©1999 Timothy G. Standish
Excipients used in biotechnology products
Solubility Enhancers:
• In general, proteins may have a tendency to aggregate and
precipitate.
• Different methods can be used to enhance solubility,
including: selection of proper pH and ionic strength conditions,
addition of amino acid or surfactants.
• Selection of appropriate enhancers is mainly dependent on:
type of protein involved and mechanism of action of the
enhancer.
©1999 Timothy G. Standish
Excipients used in biotechnology products
Anti-adsorption and anti-aggregation agents:
• Anti-adsorption agents are added to reduce
adsorption of the active protein to interfaces.
• Albumin has a strong tendency to adsorb to surfaces
and therefore added in relatively high concentrations
to protein formulation as an anti-adhesion agent.
©1999 Timothy G. Standish
Excipients used in biotechnology products
Buffer Components:
• Buffer selection is an important part of the formulation
process, because of the pH dependence of protein solubility
and physical and chemical stability.
• Buffer systems regularly encountered in biotech
formulations are phosphate, citrate and acetate.
• Even short, temporary pH changes can cause aggregation.
These conditions can occur, for example, during the freezing
step in the freeze-drying process.
©1999 Timothy G. Standish
Excipients used in biotechnology products
Preservative and Anti-oxidants:
• Methionine, cysteine, tryptophan, tyrosine and histidine are
amino acids that are readily oxidized (oxidative degradation).
• Replacement of oxygen by inert gases in the vials helps to
reduce oxidative stress. Moreover, the addition of anti-oxidants
such as ascorbic acid or sodium formaldehyde sulfoxylate.
• Certain proteins are formulated in containers designed for
multiple injection schemes. Preservatives are usually added to
minimize growth of microorganisms and thus reduce chance
for contamination.
©1999 Timothy G. Standish
Therapeutic Proteins

Insulin (diabetes)

Interferon b (relapsing MS)

Interferon g (granulomatous)

TPA (heart attack)
TPA: Tissue plasminogen activator
©1999 Timothy G. Standish
Therapeutic Proteins…







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
Infergen
©1999 Timothy G. Standish
Therapeutic Proteins…
The Problem with Proteins

Very large and unstable molecules

Structure is held together by weak noncovalent forces

Easily destroyed by relatively mild storage conditions

Easily destroyed/eliminated by the body

Hard to obtain in large quantities
©1999 Timothy G. Standish
Therapeutic Proteins…
The Problem with Proteins (in vivo)

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
©1999 Timothy G. Standish
©1999 Timothy G. Standish
Therapeutic Proteins…
The Problem with Proteins (in vitro)
Noncovalent
Covalent

Denaturation

Deamidation

Aggregation

Oxidation

Precipitation

Disulfide exchange

Adsorption

Proteolysis
©1999 Timothy G. Standish
Therapeutic Proteins…
Noncovalent Processes
Denaturation
Adsorption
©1999 Timothy G. Standish
Therapeutic Proteins…
Noncovalent Processes
Aggregation
Precipitation
©1999 Timothy G. Standish
Therapeutic Proteins…
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)
©1999 Timothy G. Standish
Therapeutic Proteins…
Deamidation
©1999 Timothy G. Standish
Therapeutic Proteins…
How to Deal with These Problems?
Storage
Formulation
Delivery
Pharmaceutics
©1999 Timothy G. Standish
Therapeutic Proteins…
Storage - Refrigeration

Low temperature reduces microbial growth and metabolism

Low temperature reduces thermal or spontaneous
denaturation

Low temperature reduces adsorption

Freezing is best for long-term storage

Freeze/Thaw can denature proteins
©1999 Timothy G. Standish
Therapeutic Proteins…
Storage - Packaging

Smooth glass walls best to reduce adsorption or precipitation

Avoid polystyrene or containers with silanyl or plasticizer
coatings

Dark, opaque walls reduce oxidation

Air-tight containers or argon atmosphere reduces air oxidation
©1999 Timothy G. Standish
Therapeutic Proteins…
Storage - Additives

Addition of stabilizing salts or ions (Zn2+ 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
©1999 Timothy G. Standish
Therapeutic Proteins…
Storage - Freeze Drying

Only cost-effective means to prepare solid, chemically
active protein

Best for long term storage

Removes a considerable amount of water from protein
lattice, so much so, that some proteins are actually
deactivated
©1999 Timothy G. Standish
Shelf Life of Protein
• Protein can be stored: as an aqueous solution, in freeze
dried form, or in dried form in a compacted state (tablet).
• Stability of protein solutions strongly depends on factors
such as pH, ionic strength, temperature and the presence of
stabilizers.
©1999 Timothy G. Standish
Shelf Life of Protein
Freeze-drying of Proteins:
• The abundant presence of large amount of water in he proteins
in solution makes it difficult to maintain preferred self life (i.e.
2 years) for protein products.
• Freeze drying may provide a good stability because of the
water removal through sublimation and not by evaporation.
• Freezing step, primary drying, secondary drying are the major
three steps in freeze drying process.
©1999 Timothy G. Standish
Therapeutic Proteins…
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%
©1999 Timothy G. Standish
Protein Pharmaceutics
Formulation
Storage
Delivery
©1999 Timothy G. Standish
Therapeutic Proteins…
The Problem with Proteins (in vivo)

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
©1999 Timothy G. Standish
Therapeutic Proteins…
Protein Formulation

Protein sequence modification (site directed mutagenisis)

PEGylation

Proteinylation

Microsphere/Nanosphere encapsulation

Formulating with permeabilizers
©1999 Timothy G. Standish
Therapeutic Proteins…
Site Directed Mutagenesis
E343H
©1999 Timothy G. Standish
Therapeutic Proteins…
Site Directed Mutagenesis

Allows amino acid substitutions at specific sites in a protein
will reduce likelihood of oxidation

Strategic placement of cysteines to produce disulfides to
increase Tm

Protein engineering (size, shape, etc.)
©1999 Timothy G. Standish
Therapeutic Proteins…
PEGylation
O
O
+
O
O
©1999 Timothy G. Standish
Therapeutic Proteins…
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
©1999 Timothy G. Standish
Therapeutic Proteins…
Proteinylation
+
Protein Drug
ScFv (antibody)
©1999 Timothy G. Standish
Therapeutic Proteins…
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
©1999 Timothy G. Standish
Therapeutic Proteins…
Microsphere Encapsulation
100 mm
©1999 Timothy G. Standish
Therapeutic Proteins…
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
©1999 Timothy G. Standish
Therapeutic Proteins…
Types of Microspheres

Nonbiodegradable
– ceramic particles
– polyethylene co-vinyl acetate
– polymethacrylic acid/PEG

Biodegradable (preferred)
– gelatin
– polylactic-co-glycolic acid (PLGA)
©1999 Timothy G. Standish
Therapeutic Proteins…
PLGA - Structure
©1999 Timothy G. Standish
Therapeutic Proteins…
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
©1999 Timothy G. Standish
Therapeutic Proteins…
Release Mechanisms
©1999 Timothy G. Standish
Therapeutic Proteins…
Peptide Micelles
©1999 Timothy G. Standish
Therapeutic Proteins…
Peptide Micelles

Small, viral sized (10-50 nm) particles

Similar to lipid micelles

Composed of peptide core (hydrophobic part) and PEG
shell (hydrophilic part)

Peptide core composition allows peptide/protein
solubilization

Also good for small molecules
©1999 Timothy G. Standish
Therapeutic Proteins…
Peptide Synthesis
©1999 Timothy G. Standish
Therapeutic Proteins…
Peptide-PEG monomers
Hydrophobic block
Hydrophilic block
Peptide
O H
H3N+
H
R1
N
H
R2 H
N
O H
PEG
O H
R3
N
H
R4
O
CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2.....
O
©1999 Timothy G. Standish
Therapeutic Proteins…
Peptide Micelles
©1999 Timothy G. Standish
Therapeutic Proteins…
Targeted Micelles
©1999 Timothy G. Standish
Therapeutic Proteins…
Nanoparticles for Vaccine Delivery to Dendritic
Cells

Dendritic Cells -‘sentries’
of the body

Eat pathogens and present
their antigens to T cells

Secret cytokines to direct
immune responses
©1999 Timothy G. Standish
Therapeutic Proteins…
Nanoparticles for Vaccine Delivery

Mimic pathogen surface characteristics

Antigen for controlled delivery within Dendritic Cells

Selective activation of cytokine genes in Dendritic Cells

Applications in Therapeutic Vaccines (e.g., cancer, AIDS, HBV,
HCV)
©1999 Timothy G. Standish
Therapeutic Proteins…
Polymeric Nanoparticle Uptake by Human DCs:
Confocal Image
©1999 Timothy G. Standish
Therapeutic Proteins…
Permeabilizers (Adjuvants)

Salicylates (aspirin)

Fatty acids

Metal chelators (EDTA)

Anything that is known to “punch holes” into the intestine
or lumen
©1999 Timothy G. Standish
Therapeutic Proteins…
Protein Formulation (Summary)

Protein sequence modification (site directed mutagenisis)

PEGylation

Proteinylation

Microsphere/Nanosphere encapsulation

Formulating with permeabilizers
©1999 Timothy G. Standish
Protein Pharmaceutics
Storage
Formulation
Delivery
©1999 Timothy G. Standish
©1999 Timothy G. Standish
Routes of Delivery

Parenteral (injection)

Oral or nasal delivery

Patch or transdermal route

Other routes
– Pulmonary
– Rectal/Vaginal
– Ocular
©1999 Timothy G. Standish
Parenteral Delivery

Intravenous

Intramuscular

Subcutaneous

Intradermal
©1999 Timothy G. Standish
Parenteral Delivery

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
©1999 Timothy G. Standish
Exubera (Inhaled Insulin)

Exubera, a dry-powder form of insulin, is
inhaled with a special device similar to an
asthma inhaler

Exubera normalized blood sugar levels as well
as injections did

Patients taking inhaled insulin also reported
greater satisfaction and quality of life (for 18+
only)

About 1/5 study subjects developed a mild
cough with inhaled insulin
Pfizer

Product pulled in Oct. 2007
©1999 Timothy G. Standish
Oral Insulin (Oralin)
©1999 Timothy G. Standish
Oral Insulin (Oralin/Oral-lyn)

Bucchal aerosol delivery system developed by Generex
(Approved in Ecaudor and India)

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
©1999 Timothy G. Standish
BioSante’s BioOral Insulin

The BioOral formulation was developed by aggregating
caseins (the principle protein in milk) around a proprietary
formulation of CAP (calcium phosphate nanoparticle),
polyethylene glycol (PEG, a polymer) and insulin by
scientists at BioSante's research center
©1999 Timothy G. Standish
Oral Delivery by Microsphere
pH 2
pH 7
©1999 Timothy G. Standish
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)
©1999 Timothy G. Standish
Patch Delivery
©1999 Timothy G. Standish
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)
©1999 Timothy G. Standish
Patch Delivery
©1999 Timothy G. Standish
GI-MAPS Layers

pH sensitive surface layer determines the adhesive site in the
GI tract

Gel-forming mucoadhesive layer adheres to GI mucosa and
permits controlled release - may also contain adjuvants

Drug containing layer holds powders, dispersions, liquids,
gels, microspheres,

Backing layer prevents attack from proteases and prevents
luminal dispersion
©1999 Timothy G. Standish
Transdermal Patches
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
Close-up of Patch Pins
©1999 Timothy G. Standish
Biocapsules
©1999 Timothy G. Standish
Summary

Protein pharmaceuticals are (and will be) the most rapidly
growing sector in the pharmaceutical repertoire

Most “cures” for difficult diseases (Alzheimers, cancer,
MS, auto-immune diseases, etc.) will probably be found
through protein drugs
©1999 Timothy G. Standish
Summary

BUT Proteins are difficult to work with

Most protein delivery is via injection

Newer methods are appearing

Oral delivery using “smart materials” is looking promising

Over the coming 3-4 years more protein drugs will have oral
formulations
©1999 Timothy G. Standish
©1999 Timothy G. Standish