advances-in-protein-chemistry

considered for their persisted existence in drug markets. These challenges are being actively tackled by intense research and development
and working on alternative technologies.
Several technologies for the production of peptides are now available which includes isolation from natural sources [45], production
by recombinant DNA technology [46], production in cell-free expression systems [47], production in transgenic animals [48] and plants
[49], production by chemical synthesis [20,50] and production by enzyme technology [51]. The type of manufacturing technologies to be
opted is primarily determined by the size of the peptide molecules. Before the advances in synthesis technologies, peptides were extracted
from natural sources for instance; human growth hormone (hGH) and insulin were collected from human corpses and slaughtered
pigs, respectively. The resultant peptides were found to be expensive, less in availability and restricted to native peptides so, alternative
methods were developed that can be applied to manufacture native as well as synthetic peptides. Even though chemical synthesis is the
most developed technology used in peptide synthesis, multi-step synthesis process, high synthesis cost, increase in length and complexity,
increasing problems with chain aggregation, low yield, high toxic effluents, and lack of specificity are severe drawbacks that can in
principle be successfully overcame by using biotechnological tools and techniques. In recent years, peptide synthesis using recombinant
DNA technology and biocatalysts has emerged as a new hope in the field of peptide drugs and their implementation can greatly affect the
cost and scalability of pharmaceutical peptides.
Recombinant DNA technology offer several advantages at large production scales and is specifically suitable for the synthesis of
large peptides and other hormones [52]. Major steps involved in recombinant production of pharmaceutical peptides includes selection
of efficient expression systems, such as Escherichia coli [53], S. aureus [54], insect cells [55], transgenic mammals [56] and transgenic
plants [57], for over expression to get larger and purer qualities of the peptides followed by fermentation, purification of fused protein,
cleavage of fused protein using endopeptidases, purification of peptide, capping of peptides (N-terminal acetylation which is in vivo
catalyzed by N-acetyltransferases or C-terminal amidation by peptidyl-glycine alpha-amidating monooxygenase (PAM)), purification of
capped peptides and formulation of final product. Although, different recombinant technologies have been developed for the production
of peptides, however, inefficient recombinant expression of peptides owing to its susceptibility to degradation in the cytoplasm mainly
due to their relatively smaller size and lack of tertiary structure, their toxicity against the bacterial host cells and post-translational
modifications particularly in case of synthesis of peptide hormones, remains a significant challenge. Direct-expression technology along
with a technology for in vivo amidation using PAM has recently been developed for the efficient and cost-effective production of peptide
hormones. To overcome the problem of degradation, peptides can be expressed in fusion with a larger protein which in turn helps
the resultant protein from proteolysis and in accumulating in cytoplasm in soluble or insoluble forms owing to their larger size [58].
However, difficulty in liberation of the fusion counterpart from the peptide by chemical or enzymatic cleavage, its purification and overall
low product yield are the major challenges associated with it that decreases the cost-effectiveness of the system. Thus, for wide application
of recombinant DNA technology, an extended and costly research and development is required as it often remains unachievable mainly
due to the less efficient expression systems and difficulties in product extraction, purification and recovery [59].
In enzymatic peptide synthesis, proteases (endo- and exo-) are the most prevalent class of enzymes that are used as biocatalyst and
are chosen based on their specificity against amino acid residues. Furthermore, it is their ability to biocatalyze reactions in non-aqueous
media such as organic solvents [60,61], supercritical fluids [62,63], eutectic mixtures [64], solid-state [65,66] and, more recently, ionic
liquids [67-70] that has expanded their applications in peptide synthesis [71,72]. In general, there are two mechanisms used in enzymecatalyzed
peptide synthesis and are based on type of the carboxyl component used, one is thermodynamically controlled synthesis (TCS)
and another is kinetically controlled synthesis (KCS) [73]. In the TCS approach, this component has a free carboxyl terminus, and the
peptide bond formation occurs under thermodynamic control. TCS is the reverse of the hydrolytic cleavage of peptide bond in presence
of proteases with the formation of a acyl intermediate, where, catalyst do not alter the equilibrium of the reaction and simply enhances
the rate of the reaction [74]. Since, the formation of acyl intermediate is the rate limiting step in TCS, the use of an acyl donor with free
carboxylic group and any type of proteases are the major considerations. The main disadvantages of this process are high requirements of
biocatalysts, need of highly précised reaction conditions to displace the equilibrium towards peptide bond formation, low reaction rates
and low product yield [75]. Displacement of equilibrium can be achieved by modifying the pH and medium composition [76,77], however
maintaining the properties of the biocatalyst, substrate and products is challenging. In the KCS approach, the carboxyl component is
used in an activated form, mainly as an ester derivative, and the synthesis occurs under kinetic control [73]. Unlike TCS, serine or
cysteine proteases (papain, thermolysin, trypsin and chymotrypsin) are used in KCS, because the main function of enzyme is to act as a
transferase destined to catalyze the transfer of an acyl group from the acyl donor to the amino acid nucleophile through the formation
of an acyl-enzyme intermediate. Enzymatically synthesized peptides not only have application in pharmaceuticals but have also been
explored in agrochemicals, human and animal nutrition [78-82]. This technology has also some limitations as it is specifically suitable
for synthesis of very small peptides [60] and cannot be used for the production of peptides more than 10 residues in length. However,
enzyme stereospecificity, mild reaction conditions, minimum side chain protection and avoidance of racemizations can overcome some
negative aspects of chemical method of peptide synthesis.
Peptide Purification and Isolation Of Purified Peptides
Before being formulated into pharmaceutically active products, the post-synthesis mixture undergoes purification and isolation
process to obtain the desired peptide product. This is most crucial step that must be carefully assessed as it plays a vital role in determining
the optimal economy of manufacturing process and eventually the utilization efficiency of peptides as therapeutic. It is a complex process
which involves several steps to ensure that the desired products meet the quality requirements set for the compound to be purified with
negligible impurities and are achieved cost-effectively. In general, the type of techniques to be used for purification purpose relies on the
properties of the peptide and the types of impurity present. Today, most commonly used standard technique for isolation is preparative
high performance liquid chromatography (HPLC) [83,84] whereas, ion-exchange chromatography, gel permeation chromatography
and medium or high pressure reversed phase chromatography are used for purification of peptides [85,86] but other methods like
counter current distribution [87] and partition chromatography [88] were used in the past. In recent years, ultra performance liquid
chromatography (UPLC) has become a technique of choice for the separation of various pharmaceutical related small organic molecules,
proteins, and peptides. Using UPLC, it is now possible to run separation process using shorter columns, and/or higher flow rates for
increased speed, with superior resolution and sensitivity [89-91].
In ion exchange chromatography (IEC), separation is dependent on the ionic interaction between the support surface and charged
groups of the peptide (cations and anions). High flow-rates, efficiency and high mechanical strength of the ion exchange material are
the most desirable factors for large-scale purification process. Low mechanical strength may have a negative impact on recovery and
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