Chapter 7

Activity and Stability of

Biological Product

Introduction
 Enzymes are protein with high molecular weight
(15000<MW<several million daltons) that act as
catalyst.
 Enzymes are specific, versatile and very effective
biological catalysts resulting in much higher reaction
rates as compared to chemically catalyzed reactions
under ambient conditions.
 Enzymes are named by adding suffix – ase to the
end of the substrate such as urease or the reaction
catalyzed such as alcohol dehydrogenase.
 Introduction
 Some protein enzymes require a nonprotein
group (cofactor, coenzyme or vitamins), for
their activity.
 Cofactor such as Mg, Zn, Fe
 Coenzyme such as NAD.

Enzyme Activity
 The activity of an enzyme may be measured by
determining the rate of product formation or substrate
used during the enzyme-catalysed reaction

 One Unit of enzyme activity is defined as that catalysing

the conversion of 1 µmol substrate (or the formation of 1
µmol product) in 1 min at 25C.

 The Enzyme Commission has recommended us of katal

(abbreviated as kat) as a new international unit of
enzyme activity.

 One kat is defined as the amount of enzyme that

transforms 1 mol of substrate per second.
Enzyme Activity
 The specific activity of an enzyme preparation is the
number of Units activity per amount of total protein
(mg protein) and is a measure of enzyme purity.

 The specific activities increases during purification

and becomes maximum and constant when the
enzyme is in a pure state.

 For example, a crude cell lysate might have a specific

activity of 0.2 units/mg protein which upon purification
may 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 pH
extremes or temperature during purification.
 The denatured enzyme will have no activity.
Enzyme Activity
 Example of calculation:

Enzyme and its activity

Factors governing catalytic activity
 The main factors determining the initial velocity of an
enzymatic reaction are enzyme concentration,
substrate concentration, pH temperature and the
presence of activators or inhibitors.
 Enzyme concentration

Factors governing catalytic activity

 Substrate concentration
Factors governing catalytic activity
 Effect of pH

Factors governing catalytic activity

 Effect of temperature
Factors governing catalytic activity
 Temperature
 The rate of enzyme-catalyzed reactions increases
with temperature up to a certain limit
 Above a certain temperature, enzyme activity
decreases with temperature because of enzyme
denaturation
 The optimum between 40-60C
Factors governing catalytic activity
Inhibition
 Certain compounds may bind to enzymes
and 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 and
reduce enzyme activity.

Factors governing catalytic activity

 Such enzyme inhibition may be reversed only
by using chelating agents such as EDTA and
citrate.
 Reversible inhibitors may dissociate more
easily from the enzyme after binding.
 Three major classes of reversible inhibitions;
competitive, noncompetitive and
uncompetitive.
Factors governing catalytic activity
 Competitive: substrate analogs and compete
with substrate for the active site of the
enzyme.
 Noncompetitive: not substrate analogs, bind
on sites other than active site and reduce
enzyme 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 was
produced (identity, strength, quality, purity) and to
deliver active ingredients at an effective level
specified during shelf-life
 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 or
chemical manipulations, including proteolytic enzymes
and heat.

Stabilizing Enzymes During

Storage
 For long-term storage, enzymes should be kept at
cryogenic temperatures in a -70°C freezer or under
liquid nitrogen
 If enzymes are stored in such a -20°C, protein stability
can be greatly enhanced by adding an equal volume of
glycerol to the sample and mixing it well. This 50%
glycerol solution will maintain the enzyme sample in the
liquid phase at 20°C
 To avoid protein denaturation during the freeze- thaw
process, it is critical that the samples be frozen quickly
and thawed quickly.
 Rapid freezing is best accomplished by immersing the
sample container in a slurry of dry ice and ethanol
 Stabilizing Enzymes During Storage
 Before the samples are frozen, they should be sterile-
filtered through a 0.22 m filter composed of a low
protein-binding material, and then placed in sterilized
cryogenic tubes to avoid bacterial contamination

 Certain additives will enhance the stability of many

enzymes for long-term storage at cryogenic
temperatures and sometimes also for short-term
storage in solution.

 Glycerol, sucrose, and cyclodextrans are often added

to stabilize enzyme

Protein Activity and Stability

 Proteins comprise an extremely heterogeneous class of
biological macromolecules.
 They are often unstable when not in their native
environments, which can vary considerably among cell
compartments and extracellular fluids.
 If certain buffer conditions are not maintained, extracted
proteins may not function properly or remain soluble.
 Proteins can lose activity as a result of proteolysis,
aggregation and suboptimal buffer conditions.
Protein Stability
 Since the biological activity of peptides and
proteins depends on the molecular
confirmation, which depends on both non-
covalent and covalent forces, these products
are extremely sensitive to changes in the
environment such as variations in
temperature, oxidation, light, ion force and
shear.

Protein storage
General Considerations for
Protein Storage
Temperature
 Generally, proteins are best stored at ≤ 4°C in clean,
autoclaved glassware or polypropylene tubes.
 Storage at room temperature often leads to protein
degradation and/or inactivity, commonly as a result of
microbial growth.
 For short term storage (1 day to a few weeks), many
proteins may be stored in simple buffers (e.g
phosphate or Tris buffers) at 4°C

General Considerations for Protein

Storage
 For long term storage for 1 month to 1 year, some
researchers choose to bead single-use aliquots of the
protein in liquid nitrogen for storage in clean plastic
containers under liquid nitrogen.

 This method involves adding the protein solution

dropwise (about 100 µl each) into a pool of liquid
nitrogen, then collecting the drop-sized frozen beads
and storing them in cryovials under liquid nitrogen
General Considerations for Protein
Storage
 Frozen at -20°C or -80°C is the more common form
of cold protein storage.
 Because freeze-thaw cycles decrease protein
stability, samples for frozen storage are best
dispensed and prepared in single-use aliquots so
that, once thawed, the protein solution will not have
to be refrozen.
 Alternatively, addition of 50% glycerol or ethylene
glycol will prevent solutions from freezing at -20°C,
enabling repeated use from a single stock without
warming (i.e., thawing).

General Considerations for Protein

Storage
Protein Concentration:
 Dilute protein solutions (< 1 mg/ml) are more prone to
inactivation and loss as a result of low-level binding
to the storage vessel.

 Therefore, it is common practice to add “carrier” or

“filler” 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.
General Considerations for Protein
Storage
Additives- to lengthen shelf life
 Cryoprotectants such as glycerol or ethylene glycol to
a final concentration of 25-50% help to stabilize
proteins by preventing the formation of ice crystals at
-20°C that destroy protein structure.
 Protease inhibitors prevent proteolytic cleavage of
proteins
 Anti-microbial agents such as sodium azide (NaN3)
at a final concentration of 0.02-0.05% (w/v) or
thimerosal 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-induced
oxidation 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 by
preventing oxidation of cysteines
 Pharmaceutical Stability
 The capability of a particular drug dosage form to
maintain its chemical, physical, and therapeutic
integrity against the microbial contamination in a
specific container closure system
 Capability of a formulation, in a specific container, to
remain 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 activity
is not less than a predetermined level of labeled
potency and its physical characteristics have not
changed appreciably or deleteriously.
Source: Remington’s Pharmaceutical Sciences
Pharmaceutical Stability
 Stability considerations will dictate the environment
for drug substance preparation and storage, choice of
packaging, and allowable shelf-life of the final drug
product.
 Should a drug substance be sensitive to
environmental factors such as temperature, humidity,
pH, light and oxygen exposure, these must be
considered and controlled when designing
processing, storage, and final packaging of the drug
product.

Pharmaceutical Stability
 For example, a light-sensitive drug will require the
minimization of exposure to certain light wavelengths
during handling and the choice of final dispensing
containers.
 Oxygen-sensitive materials will require handling
under an inert atmosphere, such as nitrogen, and the
addition of oxygen scavengers in the drug product
container.
 The reactivity of the drug substance and the
environment must be considered as well as potential
interaction of all constituents in the drug product,
excipients, and packaging.
 Parameters for stability testing
 Tablets
 Dissolution (or disintegration, if justified), water content and
hardness/friability. For coated and colour tablets additional tests
may require for texture and colour stability.
 Capsules
 Hard gelatin capsules: brittleness, dissolution (or disintegration, if
justified), 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 uniformity
per container, droplet and/or particle size distribution, weight
loss, pump delivery, microscopic evaluation (for
suspensions), foreign particulate matter and
extractable/leachable from plastic and elastomeric
components of the container, closure and pump.
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 for

clarity, homogeneity, pH, resuspendability (for
lotions), consistency, viscosity, particle size
distribution (for suspensions, when feasible), level
of microbial contamination/sterility and weight loss
(when appropriate).

Other Product Characteristics

 Visual appearance of the product (colour and opacity for

solutions/suspensions; colour, texture and dissolution time for
powders), visible particulars in solutions or after the
reconstitution of powders or lyophilised cakes, pH, and moisture
level of powders and lyophilised products.

 Sterility testing or alternatives (e.g., container/closure integrity

testing) should be performed at a minimum initially and at the
end of the proposed shelf-life.
Other Product Characteristics
 Additives (e.g., stabiliser,, preservatives) or excipients may
degrade during the dating period of the drug product.

 If there is any indication during preliminary stability studies that

reaction or degradation of such materials adversely affect the
quality of the drug product, these items may need to be
monitored 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

Minimum time period
Study Storage condition covered by data at
submission.

25  2°C/60%5 RH or 6 months (option a)

Long term 30  2°C/65% 5 RH 12 months (option b)

30  2°C/65%  5 RH 6 months
Intermediate

40  2°C/75%  5 RH 6 months
Accelerated