New and modern regulations have significantly changed the way in which therapeutic chemicals

are regulated. There is greater emphasis on the applied methodologies used to analyze and
interpret such biotech products. Those who are involved in this field equipped with a clear
understanding of where each technique or technology is used in developing and using effective,
and efficient analytical methods for characterizing and analyzing proteins, also understand the
great use of chromatography.
One area to consider in the manufacture of protein therapeutic drugs is the characteristic
structures of the components and its contribution to the properties of the product. It is a wellestablished fact that biological activity and, therefore, the therapeutic efficacy of protein drugs
depend on the exact composition and three-dimensional structure of the protein (Carr, D. 2014).
Such effects are dependent upon the nature and interaction of the molecules as well as the
resulting modification/s. These changes can manifest during initial assembly or as an
intermediate process in forming certain proteins. Recombinant proteins, for example, constitute
the fastest growing sector of the biopharmaceutical industry (Chirino and Mire-Sluis. 2004).
However, these molecules are prone to several types of post-translational modifications that can
reduce their efficacy and limit shelf life. In some cases, these modifications can also lead to
unwanted side-effects, such as triggering an immune reaction against the therapeutic protein
(De Groot. 2006). The most prevalent modifications include variable glycosylation which is the
process by which sugars are chemically attached to proteins to form glycoproteins, misfolding
and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and
proteolysis (Walsh and Jefferis. 2006). For the case of asparagine and glutamine deamidation,
residues form aspartic acid and iso-aspartic acid is another cause of protein degradation,
particularly during long-term storage (Chelius et. al. 2005). These formed deamidated
asparagine forms non-natural amino acids which are considered potentially immunogenic.
Several analytical methods based on HPLC and mass spectrometry have been developed to
detect this reaction however, residue prediction for deamidation for therapeutic proteins remain
difficult to carry out. Our recommendation is to carry out several methods and make a side-byside analysis of results to create comprehensive interpretations especially with the methods that
are complimentary in nature.
A quick look at a systematic approach to protein glycosylation analysis, requires the
understanding that this phenomenon is an important post-translational modification. It is a
feature that enhances the functional diversity of proteins and influences their biological activity
(Mario, K. et. al. 2010). These glycans are known for participation in structural roles in
molecular transport and clearance however, there functions have proven to be difficult to
understand due to the complex biosynthetic mechanisms involved. Recently, chromatographic
techniques and non-chromatography techniques (such as capillary electrophoresis and MS)
have been developed to provide better or more sensitive analysis of glycosylation patterns
(Carr, D. 2014).

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Finally, proteins can be modified with chemical reagents or with variations in chemical
conditions such as pH and thermodynamic properties like temperature. These proteins loose
tertiary structure or is denatured and the original biochemical properties are usually lost.
Additionally, aggregate formation can also occur with proteins subjected to such conditions
which also modify the collective property of the substance such as reduced activity and trigger
different immune responses once ingested. A common example for this phenomenon (although
not related to therapeutic drugs) is the changing of the physical properties of egg fluid (or
contents inside eggs) once it is subjected to high temperature. The elevated temperature in
cooking egg meals, converts the fluids (albumen and yolk) into a coagulated solid form which
now tastes different, indicating physiochemical changes took place.
For the case of protein analysis and characterization, the most powerful and useful
chromatography technique used for protein drugs is reversed-phase high performance liquid
chromatography HPLC (Carr, D. 2014). This process allows for clear and simple quantification
of therapeutic presence in a sample, with an inherent or induced chromophore, through the
detection of bound species to the utilized column. This analytical method involves the injection
of a sample into the chromatographic column which then retains the desired sample from the
mobile phase. The extracted and bound sample is increased and analyzed through a variety of
methods, which involve the detection of a chromophore of the bound agent. (Olbrich and
Corbett. 2013)
In 2002, A. R. Mehok et. al. presented a general RP-HPLC method that takes advantage of
sample displacement chromatography (SDC). The proposed method applies two isocratic steps
and renders high product purity and considerable productivity due to the high relative loadings
that are necessary in order to obtain the required sample displacement effect. The proposed
method rose from the challenge of low initial purity of the peptide samples, typical for early
development that gave rise to very challenging separations. The result of using the method
renders good purification results for small peptides at very high relative loadings without any
preceding method development (Pettersson et. al. 2004).
The separation of polypeptides by reversed-phase HPLC was initially met with problems since
dealing with large molecules proved to be impractical. Modifications were necessary in the
materials used such as the introduction of large-pore silica. These pores (150-300 angstrom)
allowed entrance of larger proteins and peptides to the adsorptive surface where the separation
process takes place. According to a paper by Vydac corporation, a 300 angstrom pore silica is
the preferred size because resolution is generally best even for small peptides. However,
smaller pore silica (60-120 angstrom) is sometimes used especially in the separation of small or
hydrophilic peptides since better resolutions are achieved in this case.
In separations, polypeptides are eluted from reversed-phase columns using aqueous mobile
phases containing an ion pairing agent and an organic modifier. It is known that Trifluoroacetic
acid is by far the most commonly used ion pairing agent because of its excellent separation
capabilities, low UV absorbance, and high volatility for easy removal in peptide isolation.
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Vydac lists organic modifiers which include acetonitrile (low viscosity, high volatility and
excellent UV absorption), isopropanol, and ethanol. It is worthwhile to note that ethanol is
commonly used in process industries which means that this compound displays sufficient
characteristics for use in large-scale applications. The reason for ethanol have such wide use
may lie in the fact that it is relatively cheap and the cost-to-benefit ratio might be low enough for
such a consideration.
Polypeptides are usually detected by UV absorption at wavelengths from 210 to 220 nm, where
the peptide bond absorbs. Higher wavelengths such as 280 nm are sometimes used to monitor
proteins with aromatic residues such as tryptophan (Pearson et. al. 1982).
Another popular method, defined by Tosoh Bioscience LLC, for the purification of proteins and
other charged molecules is ion exchange chromatography. In cation exchange chromatography
positively charged molecules are attracted to a negatively charged solid support. Conversely, in
anion exchange chromatography, negatively charged molecules are attracted to a positively
charged solid support.
The process separates proteins by charge rather than hydrophobicity. This means that
separation process that are better suited with this mechanism can perform well versus the
reversed-phase HPLC. Other conditions such as buffer pH, salt gradients, and varying pH are
also considered.
As a rule, the pH of the mobile phase buffer must be between the pI (isoelectric point) or pKa
(acid dissociation constant) of the charged molecule and the pKa of the charged group on the
solid support. A protein sample is then injected onto the column under conditions of good
retention then a gradient of linearly increasing salt concentration is then applied to elute the
sample components from the column. Varying the pH is mainly used to affect a separation
where an increased pH leads to less protonation and a decrease leads to the opposite effect.
The idea here is to make the protein no longer form ionic interactions with the positively (when
the pH is decreased) or negatively (when an increase in pH used) charged support which
eventually cause elution.
In conclusion, the processes involved in chromatography have proven to be invaluable in many
fields of study especially in the process industry. Its use in the investigation, identification, and
characterization of therapeutic drugs is so profound and essential that it has become a wellestablished subject in biochemical and process engineering discipline. Clearly no superior
method can be accurately drawn however, the nature of inquiry and substance in question plays
a major role in choosing from the available methods. For example, size exclusion
chromatography is its mild mobile phase conditions that permit the characterization of proteins
with minimal impact on the conformational structure and local environment (Fekete et. al. 2014)
however, its main disadvantage is low predictability.

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References:
A. R. Mehok et. al.; J. Chromatogr. A; 972 (2002) 87-99
Carr, D. (2014). The Role of Chromatography in the Characterization and Analysis of Protein
Therapeutic Drugs. The Column, 32(4), 24-29. Retrieved April 02, 2016, from
http://www.chromatographyonline.com/ role-chromatography-characterization-andanalysis-protein-therapeutic-drugs?id=&sk=&date=&pageID=2
Column selection guide for the separation of polypeptides by reversed-phase HPLC, Vydac
corporation. Retrieved April 02, 2016 from
http://www.seaviewsci.com/vydac/vydacpubs/handbook/hdbk1429.pdf
glycosylation. (n.d.). Collins English Dictionary - Complete & Unabridged 10th Edition. Retrieved
April 02, 2016 from Dictionary.com website
http://www.dictionary.com/browse/glycosylation
Jenkins N. Modifications of therapeutic proteins: challenges and prospects. Cytotechnology.
2007;53(1-3):121-125. doi:10.1007/s10616-007-9075-2.
Principles of Ion Exchange Chromatography. (n.d.). Retrieved April 02, 2016, from
http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/Resource
Center/PrinciplesofChromatography/IonExchange
S. Winkel Pettersson et. al.; J. Chromatogr. B; 803 (2004) 159-165
Stout, Steven J.; Dacunha, Adrian R. (1989). "Tuning and calibration in thermospray liquid
chromatography/mass spectrometry using trifluoroacetic acid cluster ions". Analytical
Chemistry.

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