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Evaluation of protein drug

delivery system
Presented by
MPHARM 1st year
Department of Pharmaceutics
Proteins are the most abundant macromolecules in
the living cells, occurring in all cells and all parts of
Cells can produce proteins that have strikingly
different properties and activities, by joining same 20
amino acids in many different combinations and
The term protein is used for molecules composed of
over 50 amino acids, and peptide for molecules
composed of less than 50 amino acids.
Proteins and peptide drugs

Management of illness through medication is

entering a new era in which a growing number of
biotechnology produced peptide and protein drugs
are available for therapeutic use.
Ailments that can be treated effectively by this new
class of therapeutic agents include cancers, memory
impairment, mental disorders, hypertension
Marketed proteins in freeze dried

Product Formulation Route Indication

Metrodin FSH 75 IU i.m. Induction of

Pergonal FSH and LH i.m. Infertility

Profasi HCG i.m. Infertility

Elspar Asparginase i.m. or i.v. Leukemia

Glucagon Glucagon i.m. or i.v. or s.c. Hypoglycemia

Acthar Corticotropin i.m. or i.v. or s.c. Hormone

Marketed peptides in ready to use
Product formulation route Indication
Pitressin 8- arginine i.m. or s.c. Post operative
Vasopressin abdominal

Lupron Leuprolide s.c. Prostatic cancer

Syntocinon Oxytocin i.m. or i.v. Labour induction

Sandostatin Octreotide s.c. Intestinal tumour

calcimar Salmon calcitonin s.c. Hypercalcemia
Structural determination of proteins

Primary structure Secondary structure Tertiary structure Quaternary structure

• Sanger’s method • CD spectroscopy • X-ray diffraction • NMR spectroscopy

• Edman’s method • FTIR spectroscopy • NMR spectroscopy
• Dansyl chloride • X-ray diffraction • Cryogenic electron
method microscopy
• Dual polarization
Evaluation of protein dds
Instrumental tests Chemical tests Biological tests
Fourier transform infrared Xanthoproteic acid test In vitro bioassay

Differential scanning Ninhydrin test In vivo bioassay


Fluorescence spectroscopy Lead sulphide test

U.V. spectroscopy Millon's test


Circular dichroism

Fluorescence resonance
energy transfer (FRET)
Fourier transform infrared spectroscopy

 applied when studying structural changes in proteins induced

by various factors and protein in different environments, and
in different sample forms, e.g. solid or in solution.
 Main advantage is the possibility of attaining information
about the secondary structure in complex systems without
any interference of the added excipients, since the
contribution from the excipients can be subtracted.
 FTIR is connected with secondary structure estimation (SSE)
software which predicts the secondary structure in seconds.
Circular dichroism

 Circular Dichroism (CD)

measures the difference
in absorbance of left
handed circularly
polarized light and left
handed circularly
polarized light.
Differential scanning calorimetry

 it is used for obtaining information on the folding

thermodynamics of globular proteins e.g. the transition from
native to denatured conformation and the unfolding of the
different domains comprising the globular protein.
 also useful for screening the effectiveness of excipients in
increasing the thermal stability.
Fluorescence spectroscopy

 Fluorescence measurements on proteins are based on the fact

that it is possible to excite the intrinsically fluorescent amino
acid residues in protein, i.e. tryptophan, tyrosine, and
phenylalanine. In complex formulations, e.g. systems
containing a liquid–liquid interface, measurements of the
changes in tryptophan fluorescence are also feasible.

 Proteins containing aromatic amino acid residues such as

phenyl alanine, tyrosine, tryptophan can be detected by u.v.
spectroscopy. Ultraviolet spectroscopy can be used for in
process quality control. Protein aggregates scatter u.v. light
and absorbance increases. Hence u.v. spectroscopy can be
used to monitor protein aggregation.
Xanthoproteic acid test

 It is an identification test of protein and it gives a positive

result with those proteins with amino acid carrying aromatic
 When protein is treated with hot concentrated nitric acid, a
yellow colored substance is formed. The yellow color is due to
xanthoproteic acid which is formed by the nitration of certain
amino acids present in protein such as tyrosine and
Ninhydrin test

This is a test for amino acids and proteins with free –

NH2 group.
When such an –NH2 group reacts with ninhydrin, an
intense blue colored complex is formed.
 Most often used technique for protein products is sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
 Proteins are denatured by boiling in the SDS solution. All charges of
protein are masked by negative charge of dodecyl sulphate. Thus
protein moves on polyacrylamide gel strictly on basis of size of
protein molecule.
 This technique is useful for determining molecular weight of
proteins. For visualization of proteins on the gel reagents are used
eg. silver nitrate, coomassie brilliant blue dye.
Fluorescence resonance energy transfer
(FRET) spectrometry
 The protein is covalently labeled with a pair of chromophores
named “donor” and “acceptor” that possess special spectral
 When the donor molecule is excited, there occurs partial transfer of
the excitation energy to the acceptor via a nonradiative dipole–
dipole coupling between the chromophores.
 The distance, typically 20–80 Å, is obtained from steady-state or
time-resolved fluorescence measurements of the extent of donor
quenching resulting from the presence of the acceptor.
In vitro bioassay

In case of in vitro bioassays response of cells to

hormones and growth factors is monitored. Human
colonic cancer cells are used to study the in vitro
effect of protein drugs.
In vivo bioassay

In case of in vivo bioassay pharmacological response

of animals to proteins is monitored : e.g., post
injection blood sugar in rabbits is monitored for
bioassay of insulin.

 Kasprzak AA, Motor Proteins Laboratory, Department of Muscle Biochemistry,

Nencki Institute of Experimental Biology, Warsaw, Poland, “The use of FRET in the
analysis of motor protein structure”.
 Jorgensen L. ∗, Moeller E.H., van de Weert M., Nielsen H.M., Frokjaer S. The Danish
University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen
O, Denmark, “Preparing and evaluating delivery systems for proteins”.
 Andreas Jabs, “Determination of Secondary Structure in Proteins by Fourier
Transform Infrared Spectroscopy”.