chapter 7 activity stability 2

8/8/2019 Chapter 7 Activity Stability 2

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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


Substrate concentration


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Effect of pH


Effect of temperature


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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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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.


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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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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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).


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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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.


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.


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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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

chapter 7 activity stability 2

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