PREFORMULATION

PREFORMULATION
PREPARED BY:-
PANDYA DEEP R.
M.PHARM SEM-1
PHARMACEUTICS
Guided By:-
Mr. Keyur Patel
Pharmaceutics Department
K.B.Raval College Of Pharmacy
1

Incompatibility- general aspects
►inactivation of drug through either decomposition or
loss of drug by its conversion to a less favorable
physical or chemical form.
►It affects safety, therapeutic efficacy, appearance or
elegance.
►When we mix two or more API and / or excipient with
each other & if they are antagonistic & affect
adversely the safety, therapeutic efficacy, appearance
or elegance then they are said to be incompatible.
2

Importance of Drug Excipient Compatibility
Study:-
 Stability of the dosage form can be maximized.
 It helps to avoid the surprise problems.
 DECS data is essential for IND (investigational
new drug ) submission.
 Determine a list of excipients that can be
used in final dosage form.
3

 Aspects of above tests are-
►Identification of compatible excipients for a
formulation
►Identification of stable storage conditions for
drug in solid or liquid state.
4

Types Of Incompatibility
 Physical incompatibility:- It involves the change in
the physical form of the formulation which involves
color changes, liquefaction, phase separation or
immiscibility.
 Chemical incompatibility:- It involves undesirable
change in formulation which is due to formation of
new chemical comp. with undesirable activity or our
formulation undergoes hydrolysis, oxidation,
reduction, precipitation, decarboxylation,
racemization.
 Therapeutic incompatibility:- It is type of in vivo
compatibility. 5

 Solid state reactions:
much slower and difficult to interpret.
 Liquid state reactions:
easier to detect - Acc. to Stability Guidelines
by FDA following conditions should be evaluated
for solutions or suspensions .
6

 FOR SOLID STATE REACTIONS:
SampleA: -mixture of drug and excipient
SampleB: -SampleA+ 5% moisture
SampleC: -Drug itself without excipients
7

 All the samples of drug-excipient blends are kept
for 1-3 weeks at specified storage conditions.
 Then sample is physically observed .
 It is then assayed by TLC or HPLC or DSC.
 Whenever feasible, the degradation product are
identified by MASS SPECTROSCOPY, NMR or
other relevant analytical techniques.
8

FOR LIQUID STATE REACTIONS:
 Place the drug in the solution of additives.
 Both flint and amber vials are used.
 This will provide information about
Susceptibility to oxidation. –
Susceptibility to light exposure. –
Susceptibility to heavy metals.
 In case of oral liquids, compatibility with ethanol,
glycerin ,sucrose, preservatives and buffers are
usually carried out. 9

Technique Name
 1) TMA a) DSC
b) DTA
 2) ASS
 3) FT-IR
 4) DRS
 5) Chromatography
 6) Radiolabelled Techniques
 7) Vapour Pressure osmometry
 8) Flourescence Spectroscopy 10

KNOWN INCOMPATIBILITIES
Functional group Incompatibility Type of reaction
Primary amine Mono & Di-saccharides Amine-Aldehyde &
Amine-Acetal
Ester, Lactone Basic component Ring opening,
Ester base hydrolysis
Aldehyde Amine, Carbohydrate Aldehyde-Amine, Schiff base
Or Glycosylamine formation
Carboxyl Base Salt formation
Alcohol Oxygen Oxidation to Aldehyde
& Ketones
Sulfhydryl Oxygen Dimerization
Phenol Metal Complexation
Gelatin- Capsule Shell Cationic Surfactant Denaturation 12

1) INTRODUCTION
Thermal method of analysis are group of
techniques in which changes in physical and /or
chemical properties of a substance are
measured as a function of temperature,
while substance is subjected to controlled
temp programme.
13

 Thermal analytical methods can measure the
following physical properties,
 a) WEIGHT LOSS ON DRYING
 b) ENTHALPY
 c) TEMP
 d) GAS EVOLUTION
 e) ELECTRICAL CONDUCTIVITY
 f) OPTICAL CHARACTERISTIC
 g) MAGNETIC PROPERTIES
 h) CHANGES IN FORM IN DIMENSION
 i) VISCOELASTIC PROPERTIES OF
SUBSTANCE
14

 They are unique methods in the field of polymer
analysis & of high value for a solid state analysis.
 They finds wide application in
 Detection of impurity
 Determination of moisture content in any drug substance
or any excipient
 Study of polymorphism
 Characterization of hydrates & solvates
 Degree of Crystallinity
 Study of phase diagram
 Drug excipient compatibility study
 Study of complexation 15

Physical properties Name of technique Instrument used
1) MASS A)THERMOGRAVIMETRY THERMO BALANCE
B)EVOLVED GAS ANALYSIS EVOLVED GAS DETECTOR
2) TEMP DIFFERENTIAL THERMAL
ANALYSIS (DTA)
DTA APPARATUS
3) ENTHALPY DIFFERENTIAL SCANNING
CALORIMETRY (DSC)
DIFFERENTIAL
CALORIMETER
4) MECHANICAL PROPERTIES
(DIMENTION,VISCOELASTIC
PROPERTIES)
A)THERMO MECHANICAL
ANALYSIS
TMA APPRATUS
B)THERMODILATOMETRY DILATOMETER
6) OPTICAL
CHARACTERISTIC
THERMOPTOMETRY
7) MAGNETIC
SUSCEPTIBILITY
THERMOMAGNETOMETRY
8) ELECTRICAL RESISTANCE
OR CONDUCTION
THERMOELECTROMETRY
16

 PRINCIPLE: TG is a technique in which a
change in the weight of a substance is recorded
as a function of temperature or time.
 Instrument: Instrument used for
thermogravimetry is thermobalance
→ Major components of a Thermobalance
 Sample container ,usually shallow platinum
crucible.
 Furnace Assembly
 Automatic recording Balance(Micro balance 17

 Factors affecting Thermogravimetry analysis are
 Heating rate
 atmosphere
 Sample characteristic
18

DATA RECORDED IN FORM OF CURVE KNOWN
AS THERMOGRAM.
THERMOGRAMS CAN BE DIVIDED INTO TWO
PORTIONS:
1) HORIZONTAL PORTION:
INDICATE REGION WHERE
THERE IS NO WEIGHT LOSS.
2) CURVED PORTION: INDICATE REGIONS OF
WEIGHT LOSS.
19

 PRINCIPLE: It is a technique in which the
energy necessary to establish a zero temp
difference between the sample & reference
material is measured as a function of temp.
 Endothermic reaction. E.g. Melting, boiling,
sublimation, vaporization, desolvation.
 Exothermic reaction: E.g crystallization,
degradation, polymerization.
20

DIFFERENTIAL SCANNING
CALORIMETRY (DSC)
 DSC is widely used to measure glass transition temp &
characterization of polymer.
 Glass Transition temp(Tg): Temp at which an
amorphous polymer or an amorphous part of
crystalline polymer goes from hard ,brittle state to
soft,
21

C) DIFFERENTIAL THERMAL ANALYSIS
(DTA)
 PRINCIPLE: A Technique in which the temperature
difference between a substance & a reference material is
measured as a function of temperature, while the substance
& reference are subjected to a controlled temperature
programme.
 The Difference in temperature is called as Differential
temp(∆t) is plotted against temperature or a function of time.
 Physical changes usually result in Endothermic peak
,whereas chemical reactions those of an oxidative nature are
exothermic.
22

MAJOR APPLICATION OF THERMAL
ANALYSIS IN PREFORMULATION
 A) Characterization of hydrates and solvates .
 B) study of polymer
 C) Detection of impurity.
 D) Prediction of stability of drug
 E) Degree of crystallinity
 F). Compatibility study of drug with excipient.
 G) Study of complexes & inclusion compounds.
23

MINOR APPLICATIONS
 - DSC is a valuable tool in choice of suppository
base.
 - In study of polymer composition ,miscibility &
individual characterization.
- Study of tablet coating
- Determinations of melting point. etc
- Determination of moisture content in drug.
- Checking technological quality grade of
disintegrate.
- Study of solid drug dispersion.
- Determination of drying temp. for different
excipients. 24

LIMITATIONS OF THERMAL ANALYSIS
 1) low sensitivity for transitions involving small
energies.
 2) The interpretation of the curves & DSC studies
should always Include several conditions & replicate.
 3) Impurity consisting of molecules of same size,
shape, & character as those of the major component
are not detected by DSC, Because these impurity fit
into the matrix of the major component without
disruption of lattice forming solid solution or inclusion
,such impurity are not detected by DSC.
 4) TGA used to studies hydrates & moisture study is
not always reliable.
25

X-RAY DIFFRACTION IN
PREFORMULATION
 METHOD:- Principle:- «- An x-ray irradiation
produce a highly specific diffraction pattern from
a crystal of material.
 An X-ray diffraction pattern from the crystal is
formed a series of dots of varying intensity with
fixed angular & recorded on photographic film. «-
 It is powerful tool for particle size analysis.
26

FTIR TECHNIQUE:
 It has been used to quantify binary
mixtures of polymorphs.
 In identification of polymorphs , only solid
samples (as mineral oil mulls & KBr pellets)
can be used .
 In solutions polymorphs of a compound have
identical spectra .
 Techniques for solid state stability studies:
 Advantages: - Rapid.
• TECHNIQUE IS QUALITATIVE & QUANTITATIVE. 27

(1) INTRODUCTION
 Impurities in pharmaceuticals are the unwanted
chemicals that remain with active pharmaceutical
ingredients (APIs),or develop during formulation, or
upon aging of APIs and formulated APIs to medicines.
 Webster‘s dictionary defines impurity are something
that is impure or makes something else impure.
.
 ►Organic volatile impurities
OVIs are generally solvents used in the synthesis or
during the formulation of the drug product.
28

 a) impurities associated in with APIs
 b) impurities that forms are created during
formulation & or with aging or that are related to
formulated forms.
 According to ICH guidelines impurities
associated with APIs are classified in to following
categories- (a)Organic impurities
(b)Inorganic impurities
(c)Residual solvents
(d)Other materials 29

Starting materials
Intermediates
Penultimate intermediate(Final
intermediates)
By-products
Interaction products
Related products
Degradation products 30

 INTERNATIONAL CONFERENCE ON HARMONISATION OF
TECHNICAL REQUIREMENT FOR REGISTRATION OF
PHARMACEUTICALS FOR HUMAN USE
 ICH HARMONISED TRIPARTITE GUIDELINE
IMPURITIES:
 GUIDELINE FOR RESIDUAL SOLVENTS
Q3C(R3) Impurities: Guideline for Residual
Solvents Q3C
 Impurities: Guideline for Residual Solvents
(Maintenance) PDE for Tetrahydrofuran (in
Q3C(R3)) Q3C(M) 31

ICH HARMONISED TRIPARTITE
GUIDELINE
 GENERAL PRINCIPLES
Classification of Residual Solvents by Risk Assessment
 The term "tolerable daily intake" (TDI) is used by
the International Program on Chemical Safety (IPCS) to
describe exposure limits of toxic chemicals and "acceptable
daily intake"(ADI) is used by the World Health Organization
(WHO) and other national and international health
authorities and institutes. The new term "permitted daily
exposure" (PDE) is defined in the present guideline as a
pharmaceutically acceptable intake of residual solvents to
avoid confusion of differing values for ADI's of the same
substance.
 Residual solvents assessed in this guideline are listed
in Appendix 1 by common names and structures. They were
evaluated for their possible risk to human health and placed
into one of three classes as follows:
32

CLASSES EVALUATED ON THE BASES OF
THEIR POSSIBLE RISK
 Class 1 solvents: Solvents to be avoided
carcinogens and environmental
e.g.,Benzene, Carben tetrachloride, 1,1-Dichlorobenzene, etc
 Class 2 solvents: Solvents to be limited
Non-genotoxic animal carcinogens or possible causative agents
of other irreversible toxicity such as neurotoxicity or
teratogenicity.
e.g.,Acetonitrile,Chlorobenzene,Chloroform,Cyclohexane
 Class 3 solvents: Solvents with low toxic potential
Solvents with low toxic potential to man;Class 3 solvents have
PDEs of 50 mg or more per day.
e.g.,Acetone, Anisol, 1-Butanol, Cumene, Ethyl ether,etc .
 Class4 solvents: Solvents for which no toxicological data
have been found
Sovents for which no adequate toxicological have been found.
e.g.,1,1-Dietoxypropane,Trifluroacetic acid,Ethyl acetate,etc.
33

METHODS FOR ESTABLISHING
EXPOSURE LIMITS:-
 The Gaylor-Kodell method of risk assessment, is
appropriate for Class 1 carcinogenic solvents. Only in cases
where reliable carcinogenicity data are available should
extrapolation by the use of mathematical models be applied
to setting exposure limits. Exposure limits for Class 1
solvents could be determined with the use of a large safety
factor
 Acceptable exposure levels in this guideline for Class 2
solvents were established by calculation of PDE values.
PDE is derived from the no-observed-effect level (NOEL),
or the lowest-observed effect level (LOEL) in the most
relevant animal study as follows:
 PDE = NOEL x Weight Adjustment
F1x F2 x F3 x F4 x F5
 The PDE is derived preferably from a NOEL. If no
NOEL is obtained, the LOEL may be used.
34

METHODS FOR ESTABLISHING
EXPOSURE LIMITS:-
 F1 = A factor to account for extrapolation between
species, F1 takes into account the comparative
surface area: body weight ratios for the species
concerned and for man.
 F2 = A factor of 10 to account for variability
between individuals.
 F3 = A variable factor to account for toxicity
studies of short-term exposure
 F4 = A factor that may be applied in cases of
severe toxicity, e.g. non-genotoxic carcinogenicity,
neurotoxicity or teratogenicity.
 F5 = A variable factor that may be applied if the
no-effect level was not established 35

LIMITS OF RESIDUAL SOLVENTS :-
 Solvent Concentration Limit (ppm)
 Benzene 2
 Carbon tetrachloride 4
 1,2-Dichloroethane 5
 1,1-Dichloroethene 8
 1,1,1-Trichloroethane 1500
Solvents to Be Avoided:-
36

LIMITS OF RESIDUAL SOLVENTS
Class 2 solvents in pharmaceutical products. .
 SOLVENT CONCENTRATION
LIMIT (PPM)
 Acetonitrile 410
 Chlorobenzene 360
Chloroform 60
 Cyclohexane 3880
 1,2-Dichloroethene 1870
Dichloromethane 600
 N,N-Dimethylacetamide 1090
 N,N-Dimethylformamide 880
 Ethylene glycol 620
 Formamide 220
 Hexane 290
 Methanol 3000
37

 Acetic acid, Heptane, Acetone,
 Isobutyl acetate, Anisole, Isopropyl acetate,
 1-Butanol, Methyl acetate, 2-Butanol,
 3-Methyl-1-butanol, Butyl acetate, Methylethyl ketone,
 tert-Butylmethyl ether, Methylisobutyl ketone, Cumene,
 2-Methyl-1-propanol, Dimethylsulfoxide, Pentane,
 Ethanol, 1-Pentanol, Ethyl acetate,
1-Propanol, Ethyl ether, 2-Propanol,
Ethyl formate, Propyl acetate Formic acid,
Class 3 solvents which should be limited by GMP or other
quality based requirements.
38

Solvents for which no adequate toxicological
data was found are given below.
o 1,1-Diethoxypropane,
o Methylisopropyl ketone,
o 1,1-Dimethoxymethane,
o Methyltetrahydrofuran 2,
o 2-Dimethoxypropane,
o Petroleum ether,
o Isooctane,
o Trichloroacetic acid,
o Isopropyl ether
39

AS PER U.S. PHARMACOPOEIA REGULATORY
LIMITS
 Organic volatile USP/NF limits USP/NF 2003
impurities before 2003 (ppm) limits (ppm)
Benzene 100 Not specific
o Chloroform 50 60
o 1,4-Dioxane 100 380
 Methylene chloride 500 600
 Trichloroethylene 100 80
40

PREFORMULATION STUDY OF
BIOTECHNOLOGICAL PRODUCTS
 INTRODUCTION TO BIOTECHNOLOGY
 "Biotechnology means any technological
application that uses biological systems, living
organisms, or derivatives thereof, to make or
modify products or processes for specific use."
41

PHYSICO-CHEMICAL PROPERTIES OF
 1. Purity, Impurities and Contaminants
 2. Structural Characterization
 3. Conformation Study
 4. Physical Characterization
42

4. PHYSICAL CHARACTERIZATION OF
 1 SOLID STATE PROPERTIES.
1 Crystallinity
2 Hygroscopicity
3 Molecular weight
4 Thermal Denaturation temperature
5 Extinction coefficient.
 2 SOLUTION PHASE PROPERTIES
1 Ionic Strength
2 pH
3 Adsorption
4 Solubility
5 Surface interaction 43

1. PURITY, IMPURITIES AND
CONTAMINANTS
 The impurities in the products are:
 Process related
cell-culture media, host cell proteins, DNA,
monoclonal antibodies
 Product related
product during the manufacture &/or storage
44

PURITY DETERMINATION
TECHNIQUE
 Protein based product
 SDS-PAGE ,HPLC ,Immunological approaches like
immunoassay
 Amino acid analysis ,N-terminal sequencing ,peptide
mapping
 Pyrogen detection
 Rabbit pyrogen test (exogenous pyrogen) and LAL
Test(endotoxins)
 DNA
 DNA Hybridization studies(dot-blot assay)utilizes
radiolabel led DNA probes
 Microbial
 virus –specific DNA probes , immunoassays.
45

2.STRUCTURAL CHARACTERIZATION
 Protein architecture are typically described in
terms of primary ,secondary ,tertiary &
quaternary & the order of stability is from
primary>>>quaternary.
 Protein structure is determined by following
techniques, SDS-gel electrophoresis ,HPLC
(mainly RP-HPLC) ,Amino acid analysis ,N-
terminal sequencing etc
46

3. CONFORMATION
 A protein’s conformation is the most elusive of its
physical properties in that it may be the most difficult
not only to ascertain but to maintain.
 One must establish the other conformational forms
present have no undesirable chemical, physical and
biological effects.
 It can be determined by the following technique ,X-ray
diffraction ( to study 3Dprotein conformation
,Circular dichrosim ,Peptide mapping ,N-terminal
sequencing or amino acid to determine amino acid
sequence. ,Spectroscopic techniques ( UV,IR, Raman,
Fluorescence)
47

4. PHYSICAL CHARACTERIZATION
SOLID STATE PROPERTIES
 Crystallinity:
 Lack of crystallinity is characterized by the low intensity
peaks or broad bands in the
 POWDER X-ray pattern.
 Other methods used are X-ray Diffraction pattern, Solid
state NMR (SS-NMR),Differential scanning calorimetry (
DSC), Thermo gravimetric analysis (TGA).
 Hygroscopicity:
 The changing environmental conditions can affect the
stability of the hygroscopic products .The estimation of
water content done by the HPLC method 48

Molecular weight:
 The molecular size of peptides and proteins are 1 to 3
orders of magnitude larger than that of conventional
drug molecules, is a major factor limiting their
diffusion across the biological membranes.
 Biopharmaceuticals molecular weight ranges from
600 to more than 10,000daltons in their non-
aggregated state
 Larger the size ,lower the permeation across
biological membranes g.i.t, ocular epithelium
,stratum corneum considered as impermeable barriers
to the permeation to the latter.
 Molecular size also restricts diffusion of the
biopharmaceutical across the polymeric barriers, used
in the fabrication of proteins and peptides delivery
systems. 49

Extinction coefficient (molar absorptivity):
 For quantification purpose, it is desirable to
measure at a particular UV-VIS wavelength for the
measurement of the protein content.
Thermal denaturation temperature:
 Most proteins can be reversibly denatured with an
increase in temperature and continued increase can
result in irreversible denaturation (seen in enzymes).
 Also in some case, protein unfolds and refolds into new
structure different then original native one.
 FREEZE –DRYING of proteins in presence of
appropriate buffer excipients can minimize the
problem
50

SOLUTION PHASE PROPERTIES
Adsorption:
 In the case of where adsorption is due to ionic
interaction of the peptide with the silanol groups on
the glass surface, can be prevented by silylating
the glass (flasks, syringes,etc) with organosilanes
such as Prosil 28.
Solubility and charge:
 The solubility of a protein is a useful indicator of
changes that may occur in the protein conformation
 If a protein denatures, more hydrophobic groups may
be exposed to the aqueous phase from the interior of
the protein.
51

PH:
 Ph has profound effect on protein stability ,folding and rates of
reaction which chemically alter the amino acid residues
 In some cases it may be possible to formulate the protein
under conditions where chemical modifications and proteolysis
are minimized as much as possible while maintaining the
protein in misfolded conformation which is readily and rapidly
refolded to “native” form upon administration.
Ionic strength:
 The concentration of counter ions is known to be an important
property in mediating electrostatic interactions in molecule,
and have large effect on stability of a protein as well as its
solubility.
 For example: Guanidium HCl is a strong naturant of protein
where as Guanidium sulphate at equivalent concentration can
stabilize proteins. 52

BIOLOGICAL
CHARACTERIZATION
 Validated biological assays should be performed at the
preformulation stage to assess the biological activity.
 For the complex molecules, the physio-chemical information may
be extensive but unable to confirm the higher order structures, a
biologic activity with a specific quantitative measure.
 Radio receptor assays (RIA) may be used to relate binding of a
protein hormone to a cellular receptor and may be used to asses
activity protein fragments, produced by the enzymatic degradation
 Immunogenicity is ability to induce the formation of antibodies when
combined with antigenicity is the ability to react with specific
antibodies can potentially result in the development of
hypersensitivity reactions.
 Techniques for minimizing the immunogenicity (considered at the
preformulation stage )
1. Cloned peptides and proteins
2. Conjugation of peptides and proteins to low molecular
weight compounds
3. Conjugation of peptides and proteins to various polymers
such as dextran, albumin, dl- amino acids, poly vinyl
pyrrolidone, polyethylene glycol,etc
53

ORAL DELIVERY OF INSULIN BY
CONJUGATION WITH VITAMIN B12
 Limitation of oral delivery of insulin:
1. Proteolysis in GIT
2. Absorption at intestinal entrocyte
 To overcome this problem, we decided to use the
dietary uptake pathway of vitamin B12. Mammals
have an active transport mechanism in the GIT for
the absorption and the cellular uptake of relatively
large vitamin B12. 54

DEGRADATION PROCESSES OF
PROTEIN PHARMACEUTICALS
 PHYSICAL INSTABILITY
1 Denaturation
2 Surface adsorption
3 Aggregation
4 Precipitation.
 CHEMICAL INSTABILITY
1 Hydrolysis
1.1 Deamidation
1.2 Intramolecular aminolysis
1.3 Transpeptidation
2 Oxidation
3 Racemization
4 β-elimination
55

METHODS TO IMPROVE
PHYSICAL STABILITY
 Additives such as metal ions ,polyalcohol
,surfactants ,reducing agents ,chelating agents
and other proteins ,amino acids ,fatty acids and
phospholipids
 Specific ion binding sites can be introduced on
protein molecules in relation to the aggregation and
precipitation. eg salts
 Both ionic and non-ionic surfactants can stabilize
proteins by preventing adsorption of proteins at
interfaces
 At low concentration in aqueous solutions
,polyhydric alcohol such as glycerol by selective
salvation .(excluding the water molecules) 56

METHODS TO IMPROVE CHEMICAL
STABILITY:
 1. Chemical modification of protein molecules:
Synthetic polymers like PEG and Polyoxyethylene
can be coupled to protein molecules chemically to
enhance the chemical stability. Comparatively, lipids
coupled with Insulin can increase the absorption of
Insulin and hence can increase the activity.
 2 Site directed mutagenesis:
This technique creates specific mutations that
alter the amino acid sequence of proteins and create
new proteins.
57

METHODS TO IMPROVE CHEMICAL
STABILITY:
 3. Appropriate choice of conditions:
 pH,
 temperature,
 preservative
 ionic strength. 58

FORMULATION APPROACHES TO
PROTEIN STABILIZATION
Protein stabilization in solution using additives:
 Concentration of salts has profound effect on protein
solution and aggregation.
 Non ionic surfactants like, polysorbate 80 inhibits
protein aggregation because of greater tendency of
surfactant molecule to align themselves at the liquid-air
interface, so, excluding the proteins from interface and
inhibiting surface denaturation.
 Use of cyclodextrin or 2-Hydroxy propyl--cyclodextrin
solubilizes ovine growth hormone and prevents shaking
induced precipitation.
59

2. Protein stabilization in the dried solid state:
Lyophilization and spray drying used to dehydrate
heat sensitive molecules and thereby inhibit the
degradative reactions that may be observed when
proteins are formulated in solution
 Mechanism for Stabilization of Dried Proteins:
 Binding of additives to the dried protein to act as a
“water substitute” after removal of hydration shell. As
water substitutes, sugars such as lactose can serve to
partially satisfy Hydrogen bonding requirements of
polar groups in dried proteins.
60

 Lyophilization and protein formulation development:
 It is characterized by two primary concerns:
 1 Excipient selection to optimize long term stability of protein in the
dried state.
 2. Effect of excipient on processing parameters of Lyophilization cycle
such as maximum and minimum drying temperature and cycle length and on
the appearance of the lyophilized powder cake.
 Freezing causes gradual concentration of solutes surrounding the protein
molecule s water is removed by the phase change.
 Spray drying of protein pharmaceuticals:
 It is alternative to freeze drying proteins as a process capable of drying
thermally labile materials.
 The particles are dried in the air stream in seconds, owing to high surface
area contact with drying gas.
 Its major advantage over Lyophilization is particle size and shape of final
dried powder. 61

GUIDELINES ON THE STABILITY OF
THE BIOTECHNOLOGICAL PRODUCT
 1 Preamble
 The applicant should develop the proper supporting
stability data for biotechnological product and
consider many external conditions that can affect the
product's potency, purity, and quality.
 The purpose of this document is to give guidance to
applicants regarding the type of stability studies that
should be provided in support of marketing
applications.
62

 2 Scope of the annex
 Stability Testing of New Drug Substances and
Products'' applies to well-characterized proteins
and polypeptides, their derivatives and products
of which they are components, and which are
isolated from tissues, body fluids, cell cultures, or
produced using recombinant deoxyribonucleic
acid (r-DNA) technology.
 The document does not cover antibiotics,
allergenic extracts, heparins, vitamins, whole
blood, or cellular blood components.
63

3 Selection of Batches
 3.1 Drug Substance (Bulk Material)
Where bulk material is to be stored after manufacture, but
before formulation and final manufacturing, stability data
should be provided on at least three batches for which
manufacture and storage are representative of the
manufacturing scale of production.
 3.2 Sample Selection
Where one product is distributed in batches differing in fill
volume (e.g., 1 milliliter (mL), 2 mL, or 10 mL), unit age
(e.g., 10 units, 20 units, or 50 units), or mass (e.g., 1
milligram (mg), 2mg, or 5 mg), samples to be entered into
the stability program may be selected on the basis of a
matrix system and/or by bracketing.
64

4. Stability-Indicating Profile
 Consequently, the manufacturer should propose a stability-
indicating profile that provides assurance that changes in
the identity, purity, and potency of the product will be
detected.
 At the time of submission, applicants should have validated
the methods that comprise the stability-indicating profile,
and the data should be available for review.
 The determination of which tests should be included will be
product-specific.
 4.1 Protocol
 The protocol should include all necessary information that
demonstrates the stability of the biotechnological product
throughout the proposed expiration dating period
including, for example, well-defined specifications and test
intervals.
65

 4.2 Potency
 For the purpose of stability testing of the
products described in this guideline, potency is
the specific ability or capacity of a product to
achieve its intended effect and is determined by a
suitable in vivo or in vitro quantitative method.
 For that purpose, a reference material calibrated
directly or indirectly against the corresponding
national or international reference material
should be included in the assay.
66

5. Storage Conditions
Temperature
 As most finished biotechnological products need
precisely defined storage temperatures, the storage
conditions for the real-time/real-temperature stability
studies may be confined to the proposed storage
temperature.
Humidity
 Biotechnological products are generally distributed in
containers protecting them against humidity.
Therefore, where it can be demonstrated that the
proposed containers (and conditions of storage) afford
sufficient protection against high and low humidity,
stability tests at different relative humidity can
usually be omitted.
67

Accelerated and Stress Conditions
 The expiration dating should be based on real-
time/real-temperature data. However it is strongly
suggested that studies be conducted on the drug
substance under accelerated and stress conditions.
 Also useful in determining whether accidental
exposures to conditions other than those proposed
(e.g., during transportation) are deleterious to the
product and also for evaluating which specific test
parameters may be the best indicators of product
stability.
 Light 68

Container/Closure
 Stability studies should include samples maintained in the
inverted or horizontal position (i.e., in contact with the
closure), as well as in the upright position, to determine the
effects of the closure on product quality.
 The applicant should demonstrate that the closure used
with a multiple-dose vial is capable of withstanding the
conditions of repeated insertions and withdrawals so that
the product retains its full potency, purity, and quality for
the maximum period specified in the instructions-for-use
on containers, packages, and/or package inserts.
Stability after Reconstitution of Freeze-Dried Product
 The stability of freeze-dried products after their
reconstitution should be demonstrated for the conditions
and the maximum storage period specified on containers,
packages, and/or package inserts. 69

6. Testing Frequency
 The shelf lives of biotechnological products may vary
from days to several years.
 With only a few exceptions, however, the shelf lives
for existing products and potential future products
will be within the range of 0.5 to 5 years.
 When shelf lives of 1 year or less are proposed, the
real-time stability studies should be conducted
monthly for the first 3 months and at 3 month
intervals thereafter.
 For products with proposed shelf lives of greater than
1 year, the studies should be conducted every 3
months during the first year of storage, every 6
months during the second year, and year of storage,
every 6 months during the second year, and annually
thereafter. 70

7. Specification:
 Although biotechnological products may be
subject to significant losses of activity,
physicochemical changes, or degradation during
storage, international and national regulations
have provided little guidance with respect to
distinct release and end of shelf life
specifications.
 Recommendations for maximum acceptable
losses of activity, limits for physicochemical
changes, or degradation during the proposed
shelf life have not been developed for individual
types or groups of biotechnological products but
are considered on a case-by-case basis.
71

8. Labeling
 For most biotechnological drug substances and
drug products, precisely defined storage
temperatures are recommended.
 Specific recommendations should be stated,
particularly for drug substances and drug
products that cannot tolerate freezing.
 These conditions, and where appropriate,
recommendations for protection against light
and/or humidity, should appear on containers,
packages, and/or package inserts.
72

QUE.
 a) A Pharmacist wants to develop a transdermal
patch of hormone. ,What test should be carried out in
order to ascertain drug-excipient incompatibility?
Give outline and significance of each test.
 Discuss the considerations of OVIs in preform
 Give an applications of Differential scanning
calorimetry and FTIR in preformulation studyulation
study.
 Why OVIs are considered in preformulation
study?
 How the preformulation studies for biotechnologically
derived products are carried out?
73

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