EVALUATION OF PROTEINS AND PEPTIDES

1. CONTENTS
1. Stability testing
2. Evaluation
3. Invitro Bioassay
4. Invivo Bioassay
5. Need of evaluation
6. Methods of evaluation

2. STABILITY TESTING
1. The capability of a particular formulation in a specific
container/closure system to remain within its physical, chemical,
microbiological , toxicological and protective specifications.
2. Stability testing refers to the evaluation of the effect of
environmental factors on the quality of the drug substance or a
formulated product which is utilized for prediction of its shelf life
and proper storage conditions.

3. EVALUATION
Many tests are required for testing the stability of protein products to
assure its identity, purity, potency and stability of formulation.
NEED OF EVALUATION
Due to complexity of proteins, bioassay are required to assess
potency of the formulation.
Bioassays are of two types: in vitro and invivo.
INVITRO BIOASSAY
In case of invitro bioassays response of cells to hormones and growth
factors is monitored.
INVIVO BIOASSAY
In case of invivo bioassay pharmacological response of animals to
proteins is monitored.

4. METHODS OF EVALUATION
1. UV Spectroscopy
2. Bradford Assay
3. Thermal analysis
4. Chromatography
5. Biuret Test
6. Electrophoresis

5. UV SPECTROSCOPY
1. It is used as an analytical determination method.
2. Proteins containing aromatic amino acid residues such as
phenyl alanine, tyrosine, tryptophan can be detected by UV
spectroscopy.
3. Ultraviolet spectroscopy can be used for in process quality
control.
4. Protein aggregates scatter u.v. light and absorbance increases.
Hence UV spectroscopy can be used to monitor protein
aggregation.

6. BARDFORD ASSAY
1. This assay employs the principle that in the presence of
proteins in an acidic medium, absorption maximum of
coomassie brilliant blue G-250 dye changes from 465nm to
595nm.
2. If there is no protein to bind, the solution remains brown.
3. The dye forms a complex with carboxyl end of proteins by
Vander Waals forces to form a blue colored solution.
4. The intensity of the colored solution can be measured using a
spectrophotometer to determine the concentration of the
protein in the sample.

7. THERMALANALYSIS
DIFFERENTIAL SCANNING CALORIMETRY
1. DSC is used to detect the glass transition temperature Tg in protein
solutions and lyophilized products.
2. It is an analysis technique used to characterize the stability of a
protein or other biomolecule directly in its native form.
3. It does this by measuring the heat change associated with the
molecule’s thermal denaturation when heated at a constant rate.
4. It can also elucidate the factors that contribute to the folding and
stability of native biomolecules. These include hydrophobic
interactions, hydrogen bonding, conformational entropy and the
physical environment.

8. 5. The precise and high quality data obtained from DSC provides vital
information on protein stability in process development, and in the
formulation of potential therapeutic candidates.
THERMOGRAVIMETRIC ANALYSIS (TGA)
It is commonly used for moisture content measurements in raw
materials and lyophilized protein products.

9. CHROMATOGRAPHY
To study stability of proteins and peptides various modes used are:
1. Normal Phase HPLC
2. Reverse Phase HPLC
3. Ion Exchange Chromatography
4. Chromatofocusing
NORMAL PHASE HPLC
1. Also known as adsorption chromatography.
2. This method separates analytes based on their affinity for a polar
stationary surface such as silica, hence it is based on analyte ability
to engage in polar interactions (such as hydrogen-bonding or dipole-
dipole type of interactions) with the sorbent surface.

10. REVERSE PHASE HPLC
1. It has become an essential tool in the separation and analysis of
proteins and peptides.
2. It is widely used in the biotechnology industry to characterize
protein therapeutic products and to analyze these for product
identity and impurities.
3. It plays a vital role in the separation of peptides from digested
proteomes prior to protein identification by mass spectrometry.
4. It is also used to purify many proteins and peptides during
investigative studies and is used for large scale purification of
protein therapeutic drugs.
5. It has a central role in protein studies because of its versatility,
sensitive detection and its ability to work together with techniques
such as mass spectrometry.

11. ION EXCHANGE CHROMATOGRAPHY
1. It discriminates between proteins on the basis of accessible surface
charges and their corresponding electrostatic interaction with the
column’s stationary phase.
2. The degree of protein retention is dependent on the strength and
number of interactions.
3. The 3-D structure of the protein determines which surface residues
will be available to contact the column’s stationary phase.
4. The net charge determines the form of IEC (anion exchange or
cation exchange) to be applied.
5. Cation exchange is used at pHs below a protein’s pI, while anion
exchange is used at pHs above a protein’s pI.
6. Protein interaction with a column’s stationary phase is dependent
on the microenvironment of the interaction site.

12. CHROMATOFOCUSING
1. It is a protein-separation technique that allows resolution of single
proteins and other ampholytes from a complex mixture according to
differences in their isoelectric points.
2. It elutes bound species by altering the pH of the buffer.
3. DRAWBACK- Proteins aggregation takes place when they are
present at relatively high concentrations and carry no net surface
charge.

13. ELECTROPHORESIS
1. Most often used technique for protein products is sodium dodecyl
sulphate polyacrylamide gelelectrophoresis (SDS-PAGE).
2. Proteins are denatured by boiling in the SDS solution. All charges of
protein are masked by negative charge of dodecyl sulphate.
3. Thus protein moves on polyacrylamide gel strictly on the basis of
size of protein molecule.
4. This technique is useful for determining molecular weight of
proteins.
5. For visualization of proteins on the gel, reagents used are
silvernitrate, coomassie brilliant blue dye.

14. EXAMPLE
 Post injection blood sugar in rabbits is monitored for
bioassay of insulin.
 It is a type of invitro bioassay.

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