Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 414

The N terminus, C terminus, alkyl groups, and other miscellaneous groups like
carbohydrates, lipids, and so on, facilitate the formation of covalent cross-links with
proteins, particularly at high temperatures. Such cross-links are mainly responsible
for loss in biological activity. The conformational structure of proteins is primarily
maintained by disulfide linkages. At alkaline pH, these intermolecular disulfide bond
interchanges lead to extensive aggregation.
�.7.2.1.� Carbamylation
Extraction of proteins produced by recombinant DNA technology or conventional
techniques requires a strong denaturant like guanidinium hydrochloride or urea. Urea
is widely used as an extractant, and such proteins are separated using ion-exchange
chromatography. However, urea contains a trace amount of cyanate that reacts with
N-terminal and leads to formation of carbamyl derivative. Carbamyl derivatives are
responsible for immunogenicity and loss of biological activity of the protein.
�.7.2.1.4 Proteolysis
Proteins undergo hydrolysis that ruptures peptide bonds at extremes of pH and high
temperature and with exposure to proteolytic enzymes. Proteolysis mainly occurs due
to bacterial contaminants. Hence, precautions taken during purification steps may be
a remedy to this problem. The addition of protease inhibitors is another alternative
approach to alleviate this problem.
8.7.2.2 Physical Alterations
�.7.2.2.1 Conformational Changes
Exposure to drastic conditions of temperature, pH, ionic strength of the media, sol-
vent characteristics, and so on, affect the conformation of proteins adversely. These
conformational changes may finally consolidate in partial or complete unfolding of
the protein, leading to loss in biological activity.
Exposure of proteins to thermodynamically unfavorable solvent systems leads to
self-association followed by aggregation and precipitation under uncontrolled con-
ditions. Aggregation is the prime step in protein degradation. The process largely
depends upon formulation conditions and hydrophobic interactions. It has also been
observed that sometimes, instead of protein aggregation, oligomers may be produced,
leading to manifestation of instability.
�.7.2.2.2 �onic Strength
Electrostatic interactions are affected by the presence of counterions. The same prop-
erty is also associated with the salting-out effect. Therefore, it becomes crucial to
maintain the ionic strength of the protein solutions and suitable buffers are incorpo-
rated to achieve the same.
�.7.2.2.� Effects of Other Formulation Components
It is necessary to screen the excipients to ensure they have no detrimental effect on
the protein bioactive. Wang and Kowal [40] have investigated a number of excipients