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Purification and polishing (Actual problems of humanity (Health issues…
Purification and polishing
purification
Chromatography
Purpose
Preparative
to purify and collect molecule of interest
collection is required
yield is important
the larger volume sample the better (millilitres to litres)
larger column diameter ( 50 – 200 mm)
increase column capacity
increase the diameter
increase the length
chromatogram less defined peaks than in analytical LC.
10 microns packed particles
sample injection is manual via a sample loop or with the aid of a simple peristaltic pump.
Analytical
compounds are diverted to waste or to a ‘destructive’ detector
mass spectrometer
charged aerosol detector
small work volumes (a few microliters or less)
high peak capacity
smaller inner diameter columns more defined peaks
column diameter 4.6 to 2.1 mm
packed with particles of 5 microns or less
backpressures can get very high
High-Pressure Liquid Chromatography
autosampler is typically used
to identify and perform qualitative and quantitative analysis of the compound
columns
monolith chromatography
column characteristics
lower flow resistance
potential for the clean-up and preparation of complex mixtures
support media for imprinting template-specific sites
applications
initially used in gas chromatography
HPLC
CEC
definition
unique porous "particle" that completely occupies the volume of the column
packed bed
stationary phase
material with high porosity
mobile phase completely circulates through the stationary phase
lower pressure drops
higher vollumetric flows
higher permeability
porosity
mesopores
high effiency
reduce the size of the macroporos and the silica skeleton
Macropor size ratio / skeleton size
Membrane chromatography
transport of solute
by convection
reducing
time
recovery volume
higher flow rate
Faster rate of mass transfer
more concentrated feed streams
promising industrial applications
proteins
enzymes
membranes
microporous
macroporous
more accessible for large
biomolecules
viruses
plasmids
large proteins
functional ligands
adsorbents
types
Electrochemical charge
cation exchange
matrix (-)
anion exchange
matrix (+)
affinity
Real affinity
very specific binding
General affinity
proteins A
Polarity
normal phase
Polar stationary phase
reverse phase
non-polar stationary phase
hidrophobic interaction
hydrophobic groups on the column
Molecular weight
size exclusion chromatography
HPLC
Most used
Research
Manufacture
Clinical tests
Diagnostics
Sample injected to column
Sample distributes
Mobile phase
Migrate faster
Stationary phase
Migrate slower
Final elution
Retention
Alkyl
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Alkyl polar embedded
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Pentafluoro-phenyl
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Cyanopropil
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Phenyl
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Aminopropyl
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Two columns
Packed
Silica microspheres
Monolithic
Porous silica
Crosslinked polymers
Scaling-Up disadvantages
Back pressure increase
Low throughput
Maintenance equipment
Resolution maintenance
Purity maintenance
Resins
Shelf life
Downside
Microorganism growth
Function loss
Solution
Preservative
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3-5 years
Electrophoresis
Identify
Quantify
Purify
DNA fragments
RNA fragments
Proteins
Types
Agarose Gel
DNA/RNA
SDS-PAGE
Proteins
2-DE
Protein Mixtures
Up to thousands
Protein/PTM abundance
Proteome analysis
Cell differentiation
Biomarker detection
Bacterial pathogenesis
Microscale protein purification
Product characterization
However
Low reproducibility
narrow dynamic range
Hydrophobic protein difficult
low throughput
Capillary
Movement of ions in electric field
Greater field strength
Faster particles move
Faster results
High resolution
Paired with MS
However
Lower robustness
Dielectrophoresis
Movement of dielectric particles
Inhomogeneous electric field
Manipulate
Discriminate
Different particle types
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polishing
final product state
enzimatic polishing
without nutrient loss
phenolics
dietary fiber
cereal grains
transformation of insoluble cell wall polysaccharides
short time
water removal
freeze drying (lyophilization)
dehydration technique
moisture removal from frozen materials by sublimation of ice from the solid phase right to the gaseous phase, omitting the liquid phase.
water steam pressure
lower than the triple-point pressure
natural environment
natural freeze-drying (freeze-out).
components
drying chamber
energy
delivery systems
desublimator
vacuum pumps
control tools
theoretical principle
the diagram "Pressure Temperature"
uses
food
freeze-dried ice cream,temperature sensitive baker`s yeast suspension and the nutrient-rich pre-boiled rice
pharmaceutical
vaccines, monoclonal antibodies,dry powders of probiotics
when stability in the liquid state is inadequate, storage requirements are too stringent, or a solid form of the product is desired
advantages
short preparation time
structure of the product stays intact
The product is of high quality
Spray drying
based on
preparation, homogenization, atomization, dispersion and subsequently dehydration of the solution
to convert liquids to powders
by spraying the feed into a hot drying medium
feeding form
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Advantages
acceptable level of degradation and oxidation of volatile compounds
imple and short process
cheaper than freeze drying
liquid products
principal problems
Wall deposition
affects the quality
and quantity of the product
affected by
operating
parameters
type and size of spray dryer
spray dryer wall properties
uses
drying of liquids which can’t be heated through their properties without a significant change of the product properties.
food
milk powder, coffee, tea, eggs, cereal, spices, flavorings, blood, starch and starch derivatives, vitamins, enzymes, stevia, nutracutical, colourings
pharmaceutical
antibiotics, medical ingredients, additives
industrial
paint pigments, ceramic materials, catalyst supports, microalgae
Vacuum Drying
large partial pressure difference between the water in the product
and the surroundings.
permits drying
at a lower temperature
very important for
products that may suffer significant flavor changes at higher temperatures
other polishing techniques
gel permeation chromatography
dyalisis
crystalization
Parameters to choose the best purification and polishing technique
purity
activity
physical and structural properties
compound of interest
matrix
stabilizing conditions
temperature
pH
pressure
application
Problems of scaling-up
Keeping the same parameters
Changes in chemical dynamics
Substrate and products concentration
Nutrients
Micronutrients and stabilizing agents
Temperature
pH
Changes in transport phenomena
Mixing
Power input
Shear induced by agitation
Need for screening biochemical parameters and growth kinetics
Adequate process design for physical parameters
For parameters that are known to have an effect on the biological agent
Oxygen supply
Heat transfer and mixing
Aeration
Agitation
Mixing time
Power input
Oxygen mass transfer coefficient
Keeping the same
Applications of scaling-down
Investigation of manufacturing deviations
Raw material investigations
Evaluation and qualification of new GMPs
Enhancing process understanding
Process characterization studies
Improving process robustness
Optimization of parameters
Actual problems of humanity
Health issues
Sickness
Malaria
Diarrhoea
Exposure to indoor smoke from solid fuels
Exposure to urban air pollution
Unintentional poisonings
Disruption of life-sustaining ecosystems
Overpopulation
Environmental degradation
Intensification of harmful agricultural practices
Industrialization
Industrial pollution
Production of toxic wastes
Petroleum wastes
Lubricants
Dielectric fluids
Flame retardants
Refrigerants
Heat transfer fluids
Increasing energy use
Water pollution
Widespread poverty
Lack of public infrastructure
Lack of healthcare
Climate change
Rise of world temperature
Greenhouse gases emissions
Depletion of stratospheric ozone
Loss of biological diversity
Deforestation
Desertification
Acid precipitation
Major epidemics of people and crops
Threat of nuclear war
World hunger
Biotechnological solutions
Remediation of hazardous waste sites
Engineering applications of aerobic metabolism
Optimizing the concentrations of oxygen and nutrients
Assuring adequate moisture in an environment
Enhancing of indigenous microbial population
Genetic enhancement of biodegradation
Slurry-phase bioremediation of contaminated soil
Use of microorganisms for biofermenation
Adaptation and specificity of local floral to degrade waste
Drug discovery and development
Cancer
Central nervous system
Angiogenesis
Infectious disease
Assay development and screening
Toxicological screening
Food security
Production of nutritionally enhanced crops
Constraints for biological solutions
Acceptance of new technologies is slow
Concern over possible negative side effects
Religious beliefs
Indifference
Strict regulations
Transgenic crops
Poor communication by the scientific community
Biotechnology and medicine
New methods to treat diseases
Gene therapy
Production of drugs and treatments
Vaccines
Antibiotics
Hormones
Recombinant drugs
Blood factors
Growth factors
Cytokines
Enzymes
Monoclonal antibodies
Knowledge of genetic basis of heritable diseases
Invention of technology to manipulate and fix mutant genes
Testing for suspected genetic defects for optimal treatments
Knowledge of the probability of developing disease
Detection of disease in fetuses and embryos
Biotechnology and Ecology
Biofuel production
Ethanol production
Biodiesel
Biomethane
Biogas production
Petroleum upgrading
Removal of undesirable elements/components
Crop improvement
Bioremediation
Microbiologically influenced corrosion
Recycling organic waste
Synthetic biology
Cell-free synthetic biology
Activation of biological machinery without the use of living cells
Transcription
Translation
Metabolism
Cell-free systems
Extract-based systems
Basic transcription and translation functions
DNA templates
Energy regeneration substrates
Amino acids
Nucleotides
Cofactors
Salts
Purified system
Toolbox of purified E. coli translational components
Synthetic enzymatic pathway system
Numerous enzymes for implementing complicated bioreactions
Excipients
Objectives
Bulk up the formulation
Improve patient acceptance
Protect, support and enhance stability of a formulation
Improve bioavailability of active drug
Enhance overall safety and effectiveness
Definition
Any substance other than active drug or pro-drug that is included in the manufacturing process
Classification
Based on their functions
Anesthetics
Laxatives
Solvents
pH modifiers
Astringent
Carminative
Based on their origin
Vegetable source
Starch
Mineral source
Calcium phosphate
Animal source
Gelatin
Synthetic
Boric acid
Functions
Diluents
Lubricants
Binders
Disintegrating agent's
Antimicrobial agents
Selection is based on
Material consistency
Regulatory acceptance
Functionality
Cost
Sources
It must be:
Non reactive
Inert to human body
Chemically stable
Type of interactions
Excipient-Excipient Interaction
Co processed excipients
These excipient offer in enhancing various properties like dissolution and absorption
Aimed at improving flow properties
Added functionality mannitol for orally disintegrating tables
Used because of its property to prepare robust tablets
Principle
Though observed very rarely, these are of prime importance in determining the stability of the dosage forms
Added functionality partially pregelatinized starches
They are used as fillers in hard gelatin capsules binders
Package-Excipient Interaction
Most commonly used packaging and interactions
Plastic
Migration
Moisture uptake
Leaching
Metals
Reactivity
Corrosion
Glass
Leaching of alkali
Oxidative reactions
Rubber
Leaching
Water permeability
Package
Should preserve the integrity of the products
Should be inert in nature
Should protect the product from environmental conditions
Drug-Excipient Interaction
Principle
Excipients can affect the active ingredient and lead to formation of molecular complexes
The active pharmaceutical ingredients are in intimate contact with the excipients which are in greater quantity
Classification
Physical interactions
Interactions
Solid dispersion
Improves the dissolution and bioavailability of hydriphobic drugs
Adsorption
Can lead to reduced bioavailability as the drug in not available for dissolution
Complexation
Binds reversibly with drugs to form insoluble complexes
Difficult to detect
Permit the components in the formulation to retain their molecular structure
Physical interactions alter the rate of dissolution
Biopharmaceutical interactions
Interactions
Premature breakdown of enteric coat
Increase in gastrointestinal motility
Interactions due to adjunct therapy
Interactions between the medicine and the body fluids will have an influence in the rate absorption
This interaction is observed after administration of the medication
Chemical interactions
Principle
They have a deleterious effect on the formulation
Active pharmaceutical ingredients and excipients react with each other to form unstable compounds
Interactions
Photolysis
Exposure to light may lead to discoloration or even decomposition of product
Polymerization
The polymorphic forms possess higher potential energy
Racemization
Conversion of a chemical into its optical isomer
Hydrolysis
Drugs with functional groups
Oxidation
Oxidative reaction are catalyzed by oxygen
Protein aggregations
Is tied to protein folding, stability and molecular chaperones
Classification
Ordered
Examples
Amyloid fibrils
Amyloid plaque
Fibrous morphology
X-ray fiber diffraction
Characteristic birefringent
Disordered
Examples
In vivo inclusion bodies
Major problem in biotechnology and development of protein drugs
They arise from native protein of very limited solubility
In vitro aggregates are those formed during the refolding of unfolded proteins
Can result in
Amyloid fibrils
Formation of inclusion bodies
Folding aggregates
Can cause major economic and technical problems in the biotechnology and pharmaceutical industry
Conditions of aggregation
Isoelectric precipitation
Salting out
Problems with protein aggregation
Protein deposition diseases
Transmissible spongiform encephalopathies
Alzheimer's disease
BSE
Inclusion bodies
Renaturation is frequently difficult
Protein drugs
Protein aggregation can occur during lyophilization
Factors favoring aggregation
Mutations
Environmental conditions
Depends on
The protein amino acid sequence
pH
Temperature
Kinetic competition
