chapter 7 activity stability 2
TRANSCRIPT
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Chapter 7
Ac t iv it y and St ab i l i t y o f
B io log ic a l Produc t
Introduction
Enzymes are protein with high molecular weight
(15000
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Introduction
Some protein enzymes require a nonprotein
group (cofactor, coenzyme or vitamins), fortheir activity.
Cofactor such as Mg, Zn, Fe
Coenzyme such as NAD.
Enzyme Activity
The activity of an enzyme may be measured bydetermining the rate of product formation or substrateused during the enzyme-catalysed reaction
One Unit of enzyme activity is defined as that catalysingthe conversion of 1 mol substrate (or the formation of 1mol product) in 1 min at 25C.
The Enzyme Commission has recommended us of katal(abbreviated as kat) as a new international unit ofenzyme activity.
One kat is defined as the amount of enzyme thattransforms 1 mol of substrate per second.
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Enzyme Activity
The specific activity of an enzyme preparation is thenumber of Units activity per amount of total protein(mg protein) and is a measure of enzyme purity.
The specific activities increases during purificationand becomes maximum and constant when theenzyme is in a pure state.
For example, a crude cell lysate might have a specific
activity of 0.2 units/mg protein which upon purificationmay increase to 10 units/mg protein.
Enzyme Activity
Only enzyme that remains catalytically active
will be measured.
The enzyme may be denatured if it unfolds or
has its three dimensional shape altered by pHextremes or temperature during purification.
The denatured enzyme will have no activity.
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Enzyme Activity
Example of calculation:
Enzyme and its activity
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Factors governing catalytic activity
The main factors determining the initial velocity of an
enzymatic reaction are enzyme concentration,substrate concentration, pH temperature and thepresence of activators or inhibitors.
Enzyme concentration
Factors governing catalytic activity
Substrate concentration
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Factors governing catalytic activity
Effect of pH
Factors governing catalytic activity
Effect of temperature
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Factors governing catalytic activity
Temperature
The rate of enzyme-catalyzed reactions increases
with temperature up to a certain limit
Above a certain temperature, enzyme activitydecreases with temperature because of enzymedenaturation
The optimum between 40-60C
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Factors governing catalytic activity
Inhibition
Certain compounds may bind to enzymesand reduce their activity. These compounds
are known as enzyme inhibitors.
Enzyme inhibitions may be irreversible or
reversible.
Irreversible inhibitors such as heavy metal
form a stable complex with enzyme andreduce enzyme activity.
Factors governing catalytic activity
Such enzyme inhibition may be reversed only
by using chelating agents such as EDTA andcitrate.
Reversible inhibitors may dissociate moreeasily from the enzyme after binding.
Three major classes of reversible inhibitions;
competitive, noncompetitive anduncompetitive.
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Factors governing catalytic activity
Competitive: substrate analogs and compete
with substrate for the active site of theenzyme.
Noncompetitive: not substrate analogs, bindon sites other than active site and reduceenzyme affinity to the substrate.
Uncompetitive: bind to the ES complex only
and have no affinity for the enzyme itself.
STABILITY
The ability of a product to remain in compliance with
its established specification to be the same as it wasproduced (identity, strength, quality, purity) and todeliver active ingredients at an effective levelspecified during shelf-life
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Enzyme stability
The extent to which an enzyme retains its structural
conformation or its activity when subjected to storage,isolation, and purification or various other physical orchemical manipulations, including proteolytic enzymesand heat.
Stabilizing Enzymes During
Storage For long-term storage, enzymes should be kept at
cryogenic temperatures in a -70C freezer or underliquid nitrogen
If enzymes are stored in such a -20C, protein stabilitycan be greatly enhanced by adding an equal volume ofglycerol to the sample and mixing it well. This 50%glycerol solution will maintain the enzyme sample in the
liquid phase at 20C
To avoid protein denaturation during the freeze- thawprocess, it is critical that the samples be frozen quicklyand thawed quickly.
Rapid freezing is best accomplished by immersing thesample container in a slurry of dry ice and ethanol
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Protein Stability
Since the biological activity of peptides and
proteins depends on the molecularconfirmation, which depends on both non-covalent and covalent forces, these products
are extremely sensitive to changes in theenvironment such as variations intemperature, oxidation, light, ion force and
shear.
Protein storage
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General Considerations for
Protein StorageTemperature
Generally, proteins are best stored at 4C in clean,
autoclaved glassware or polypropylene tubes.
Storage at room temperature often leads to proteindegradation and/or inactivity, commonly as a result ofmicrobial growth.
For short term storage (1 day to a few weeks), manyproteins may be stored in simple buffers (e.g
phosphate or Tris buffers) at 4C
General Considerations for Protein
Storage
For long term storage for 1 month to 1 year, some
researchers choose to bead single-use aliquots of theprotein in liquid nitrogen for storage in clean plasticcontainers under liquid nitrogen.
This method involves adding the protein solutiondropwise (about 100 l each) into a pool of liquidnitrogen, then collecting the drop-sized frozen beads
and storing them in cryovials under liquid nitrogen
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General Considerations for Protein
Storage Frozen at -20C or -80C is the more common form
of cold protein storage.
Because freeze-thaw cycles decrease proteinstability, samples for frozen storage are bestdispensed and prepared in single-use aliquots sothat, once thawed, the protein solution will not haveto be refrozen.
Alternatively, addition of 50% glycerol or ethylene
glycol will prevent solutions from freezing at -20C,enabling repeated use from a single stock withoutwarming (i.e., thawing).
General Considerations for Protein
Storage
Protein Concentration:
Dilute protein solutions (< 1 mg/ml) are more prone toinactivation and loss as a result of low-level binding
to the storage vessel.
Therefore, it is common practice to add carrier orfiller protein, such as purified bovine serum albumin(BSA) to 1-5 mg/ml (0.1-0.5%), to dilute protein
solutions to protect against such degradation and
loss.
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General Considerations for Protein
StorageAdditives- to lengthen shelf life
Cryoprotectants such as glycerol or ethylene glycol to
a final concentration of 25-50% help to stabilizeproteins by preventing the formation of ice crystals at-20C that destroy protein structure.
Protease inhibitors prevent proteolytic cleavage ofproteins
Anti-microbial agents such as sodium azide (NaN3)
at a final concentration of 0.02-0.05% (w/v) orthimerosal at a final concentration of 0.01 % (w/v)
inhibit microbial growth.
General Considerations for Protein
Storage
Metal chelators such as EDTA at a final
concentration of 1-5 mM avoid metal-inducedoxidation of SH groups and helps to
maintain the protein in a reduced state. Reducing agents such a dithiothreitol (DTT)
and 2-mercaptoethanol (2-ME) at final
concentrations of 1-5 mM also help to
maintain the protein in the reduced state bypreventing oxidation of cysteines
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Pharmaceutical Stability The capability of a particular drug dosage form to
maintain its chemical, physical, and therapeuticintegrity against the microbial contamination in aspecific container closure system
Capability of a formulation, in a specific container, toremain within its physical, chemical, microbiological,therapeutic, and toxicological specifications.
The time from the date of manufacture and packaging
of the formulation until its chemical or biological activityis not less than a predetermined level of labeledpotency and its physical characteristics have notchanged appreciably or deleteriously.
Source: Remingtons Pharmaceutical Sciences
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Pharmaceutical Stability Stability considerations will dictate the environment
for drug substance preparation and storage, choice ofpackaging, and allowable shelf-life of the final drugproduct.
Should a drug substance be sensitive toenvironmental factors such as temperature, humidity,pH, light and oxygen exposure, these must beconsidered and controlled when designingprocessing, storage, and final packaging of the drug
product.
Pharmaceutical Stability
For example, a light-sensitive drug will require theminimization of exposure to certain light wavelengthsduring handling and the choice of final dispensingcontainers.
Oxygen-sensitive materials will require handlingunder an inert atmosphere, such as nitrogen, and theaddition of oxygen scavengers in the drug productcontainer.
The reactivity of the drug substance and theenvironment must be considered as well as potentialinteraction of all constituents in the drug product,excipients, and packaging.
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Parameters for stability testing
Tablets
Dissolution (or disintegration, if justified), water content andhardness/friability. For coated and colour tablets additional testsmay require for texture and colour stability.
Capsules
Hard gelatin capsules: brittleness, dissolution (or disintegration, ifjustified), water content, and level of microbial contamination.
Emulsions
Phase separation, pH, viscosity, level of microbial contamination,
and mean size and distribution of dispersed globules. Oral solutions and suspensions
Formation of precipitate, clarity for solutions, pH, viscosity,extractables, level of microbial contamination.
Parameters for stability testing
Powders and granules for oral solution or suspension
Water content, and reconstitution time.
Nasal sprays: solutions and suspensions
Clarity (for solution), level of microbial contamination, pH,particulate matter, unit spray medication content uniformity,number of actuations meeting unit spray content uniformityper container, droplet and/or particle size distribution, weightloss, pump delivery, microscopic evaluation (forsuspensions), foreign particulate matter andextractable/leachable from plastic and elastomericcomponents of the container, closure and pump.
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Parameters for stability testing
Topical preparations
Included in this broad category are ointments,
creams, lotions, paste, gel, solutions, eye drops,and sprays.
Topical preparations should be evaluated forclarity, homogeneity, pH, resuspendability (forlotions), consistency, viscosity, particle size
distribution (for suspensions, when feasible), levelof microbial contamination/sterility and weight loss
(when appropriate).
Other Product Characteristics
Visual appearance of the product (colour and opacity forsolutions/suspensions; colour, texture and dissolution time forpowders), visible particulars in solutions or after thereconstitution of powders or lyophilised cakes, pH, and moisturelevel of powders and lyophilised products.
Sterility testing or alternatives (e.g., container/closure integritytesting) should be performed at a minimum initially and at theend of the proposed shelf-life.
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Other Product Characteristics
Additives (e.g., stabiliser,, preservatives) or excipients maydegrade during the dating period of the drug product.
If there is any indication during preliminary stability studies thatreaction or degradation of such materials adversely affect thequality of the drug product, these items may need to bemonitored during the stability program.
The container/closure has the potential to adversely affect the
product and should be carefully evaluated (see below).
Storage condition for stability study
Study Storage condition
Minimum time periodcovered by data atsubmission.
Long term
25 2C/60%5 RH or30 2C/65% 5 RH
6 months (option a)12 months (option b)
Intermediate30 2C/65% 5 RH 6 months
Accelerated40 2C/75% 5 RH 6 months