Literature Review - The Role of Chromatography in the Characterization and Analysis of Protein Therapeutic Drugs by C. David Carr

8/18/2019 Literature Review - The Role of Chromatography in the Characterization and Analysis of Protein Therapeutic Drug…

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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 well-

established fact that biological activity and, therefore, the therapeutic efficacy of protein drugs

depend on the e act composition and three-dimensional structure of the protein !"arr, #. $%&'(.

)uch 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. +ecombinant proteins, for e ample, constitute

the fastest growing sector of the biopharmaceutical industry !"hirino and ire-)luis. $%%'(.

owever, 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

!#e root. $%%/(. 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, o idation of methionine, deamidation of asparagine and glutamine, and

proteolysis !0alsh and 1efferis. $%%/(. 2or 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 !"helius et. al. $%%3(. These formed deamidated

asparagine forms non-natural amino acids which are considered potentially immunogenic.

)everal analytical methods based on 45" 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 ma6e a side-by-

side analysis of results to create comprehensive interpretations especially with the methods that

are complimentary in nature.

7 quic6 loo6 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

! ari8o, 9. et. al. $%&%(. These :glycans; are 6nown for participation in structural roles in

molecular transport and clearance however, there functions have proven to be difficult to

understand due to the comple biosynthetic mechanisms involved. +ecently, chromatographictechniques and non-chromatography techniques !such as capillary electrophoresis and )(

have been developed to provide better or more sensitive analysis of glycosylation patterns

!"arr, #. $%&'(.

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2inally, proteins can be modified with chemical reagents or with variations in chemical

conditions such as p and thermodynamic properties li6e temperature. These proteins loose

tertiary structure or is ected to such conditions

which also modify the collective property of the substance such as reduced activity and trigger different immune responses once ingested. 7 common e ample 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 sub>ected to high temperature. The elevated temperature in

coo6ing egg meals, converts the fluids !albumen and yol6( into a coagulated solid form which

now tastes different, indicating physiochemical changes too6 place.

2or the case of protein analysis and characterization, the most powerful and useful

chromatography technique used for protein drugs is reversed-phase high performance liquidchromatography 45" !"arr, #. $%&'(. 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 in>ection

of a sample into the chromatographic column which then retains the desired sample from the

mobile phase. The e tracted 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

"orbett. $%&?(

In $%%$, 7. +. eho6 et. al. presented a general +4- 45" method that ta6es advantage of

sample displacement chromatography !)#"(. 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 !4ettersson et. al. $%%'(.

The separation of polypeptides by reversed-phase 45" was initially met with problems since

dealing with large molecules proved to be impractical. odifications were necessary in the

materials used such as the introduction of large-pore silica. These pores !&3%-?%% angstrom(

allowed entrance of larger proteins and peptides to the adsorptive surface where the separation

process ta6es place. 7ccording to a paper by @ydacA corporation, a ?%% angstrom pore silica is

the preferred size because resolution is generally best even for small peptides. owever,

smaller pore silica !/%-&$% 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 6nown that Trifluoroacetic

acid is by far the most commonly used ion pairing agent because of its e cellent separation

capabilities, low B@ absorbance, and high volatility for easy removal in peptide isolation.

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@ydacA lists organic modifiers which include acetonitrile !low viscosity, high volatility and

e cellent B@ 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.

4olypeptides are usually detected by B@ absorption at wavelengths from $&% to $$% nm, where

the peptide bond absorbs. igher wavelengths such as $C% nm are sometimes used to monitor

proteins with aromatic residues such as tryptophan !4earson et. al. &DC$(.

7nother popular method, defined by Tosoh Eioscience 55", for the purification of proteins and

other charged molecules is ion e change chromatography. In cation e change chromatography

positively charged molecules are attracted to a negatively charged solid support. "onversely, in

anion e change 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 45". Other conditions such as buffer p , salt gradients, and varying p are

also considered.

7s a rule, the p of the mobile phase buffer must be between the pI !isoelectric point( or p9a

!acid dissociation constant( of the charged molecule and the p9a of the charged group on the

solid support. 7 protein sample is then in>ected 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. @arying the p is mainly used to affect a separation

where an increased p leads to less protonation and a decrease leads to the opposite effect.

The idea here is to ma6e the protein no longer form ionic interactions with the positively !when

the p is decreased( or negatively !when an increase in p used( charged support whicheventually 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 well-

established sub>ect in biochemical and process engineering discipline. "learly no superior

method can be accurately drawn however, the nature of inquiry and substance in question plays

a ma>or role in choosing from the available methods. 2or e ample, size e clusion

chromatography is its mild mobile phase conditions that permit the characterization of proteins

with minimal impact on the conformational structure and local environment !2e6ete et. al. $%&'(

however, its main disadvantage is low predictability.

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+eferencesF

7. +. eho6 et. al.G 1. "hromatogr. 7G DH$ !$%%$( CH-DD

"arr, #. !$%&'(. The +ole of "hromatography in the "haracterization and 7nalysis of 4rotein

Therapeutic #rugs. The Column, 32 !'(, $'-$D. +etrieved 7pril %$, $%&/, from

httpF**www.chromatographyonline.com* role-chromatography-characterization-and-

analysis-protein-therapeutic-drugs idJKs6JKdateJKpageI#J$

"olumn selection guide for the separation of polypeptides by reversed-phase 45", @ydac

corporation. +etrieved 7pril %$, $%&/ from

httpF**www.seaviewsci.com*vydac*vydacpubs*handboo6*hdb6&'$D.pdf

glycosylation. !n.d.(. Collins English Dictionary - Complete & Unabridged 10th Edition . +etrieved

7pril %$, $%&/ from #ictionary.com website

httpF**www.dictionary.com*browse*glycosylation

1en6ins N. odifications of therapeutic proteinsF challenges and prospects. Cytotechnology .

$%%HG3?!&-?(F&$&-&$3. doiF&%.&%%H*s&%/&/-%%H-D%H3-$.

4rinciples of Ion L change "hromatography. !n.d.(. +etrieved 7pril %$, $%&/, from

httpF**www.separations.us.tosohbioscience.com*)ervice)upport*Tech)upport*+esource

"enter*4rinciplesof"hromatography*IonL change

). 0in6el 4ettersson et. al.G 1. "hromatogr. EG C%? !$%%'( &3D-&/3

tout, te!en "#$ Dacunha, %drian # '1()(*# +Tuning and calibration in thermospray li uid

chromatography mass spectrometry using tri.luoroacetic acid cluster ions+# %nalytical

Chemistry#

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Literature Review - The Role of Chromatography in the Characterization and Analysis of Protein Therapeutic Drugs by C. David Carr