|
|
|
European Biopharmaceutical Review
|
The increased complexity of protein therapeutic drugs, as well as
ever-increasing regulatory requirements, has demanded improved
analytical tools for protein identification. Advances in HPLC technology
have brought improved speed, sensitivity and quantification of protein
and peptide therapeutics.
The physical analysis of biopharmaceutical drugs over the last decade
has seen dramatic changes due to advances in analytical technologies, as
well as improved methodologies for analysing protein therapeutics.
While the 1990s saw advances in sensitivity and throughput due to
innovations in detection technologies such as mass spectrometry, the
last decade has been more influenced by advances upstream of the mass
spectrometer with significant improvement in both HPLC instrumentation
as well as column technology. New HPLC instrumentation features very low
system volumes and high performance specifications. Such improvements
minimise the loss of chromatographic performance due to instrumentation
limitations, with extra column system volume standing out as the most
negative influence on separations. Higher performing HPLC systems (often
called UHPLC) maximise the performance of next-generation UHPLC
columns, which demonstrate improved resolution and higher peak capacity
versus older 1980s-era protein columns (1,2).
For protein therapeutics this new paradigm of reversed phase
chromatography has been especially helpful. Second generation protein
therapeutics are typically larger than the first generation, generating
more complex separations, especially for peptide map applications where
large proteins can generate hundreds of peptide peaks demanding
separation. Modern protein therapeutics are often Immunoglobulin G based
(IgG), an extraordinarily complex protein with multiple types of post
translational modifications (PTMs) (deamidation, glycosylation,
glycation, and so on) that require maximum chromatographic separation to
quantify any minor species present (3). Additional analytical
challenges for modern protein therapeutics include PEGylation, where
large poly-disperse polyethylene glycol is added to a protein or peptide
to increase in vivo half-life and improve bioavailability. While such
modifications improve the drug ability of a protein, from an analytical
characterisation point of view such modification makes characterisation
extremely difficult; this is right at a time where regulatory agencies
require increasing amounts of information on protein therapeutics.
Adaptation of UHPLC column technologies to protein and peptides comes at
an opportune time to address the increasing analytical requirements for
protein therapeutics. With the development of small, fully-porous
type-B (low metal) silica particle columns, as well as improved
instrumentation, increased resolution and throughput has been realised
for reversed phase separation of small molecules and peptides. A further
development in the last three years has been the introduction of
geometrically structured silica particle columns (also known as
core-shell, superficially porous, or Poroshell) that have provided equal
or better chromatographic performance for peptide and peptide mapping
applications without pressure and instrumentation limitations of small
particle, fully-porous media (see Figure 1) (4). Most recently we have
seen the introduction of peptide applicationspecific and protein
application-specific core-shell media specifically designed for the
difficulties in analysing proteins and peptides. Surprisingly,
separation principles and operating parameters for separating proteins
and peptides are significantly different despite both being polymers of
amino acids (proteins being considered greater than 10KDa molecular
weight versus peptides which are less than 10KDa) (5). Key to successful
method development for these separations is understanding the
requirements for each application.
Peptide Mapping and Peptide Separations
The primary application that analytical biochemists perform to
characterise proteins is called tryptic, enzymatic or peptide mapping,
where a large protein is digested with a proteolytic enzyme into smaller
peptide fragments. This mixture of peptides is chromatographically
separated by a reversed phase HPLC column and individual peptides are
positively identified by tandem mass spectrometry. Peptide mapping
allows for identification and quantification of very low level
impurities and post-translational modifications present in a
biotherapeutics sample, provided that the peptide can be
chromatographically separated from other peptides in the mixture. For
firstgeneration protein therapeutics, which were typically smaller in
size and less complex, separating the small number of peptide fragments
generated in a peptide map was feasible with older column technologies.
However, large protein therapeutics like IgG monoclonal antibodies can
generate hundreds of peptide fragments, making resolution of PTM
indicating peptides difficult using low-resolving HPLC methods.
Recent UHPLC column technologies, such as sub-2μm porous media and
core-shell media columns, provide substantial benefit for analysing
complex peptide maps. An example of the improvement that new UHPLC
columns can provide for peptide mapping applications is shown in Figure
2, where significant increases in peak capacity and separation are
realised by using a core-shell peptide mapping column versus older fully
porous 3μm or 5μm columns. Key reasons for performance improvement
relates to understanding the behaviour of peptides and how column
parameters influence separations. Peptide retention of less than 10KDa
is primarily influenced by the chemical composition of the amino acids
that make up the peptide chain (charged, polar or non-polar). Many
peptides are moderately polar with only minor differences in
hydrophobicity, thus a fairly hydrophobic stationary phase with high
methylene selectivity is necessary to separate chemically similar
peptides. High efficiency media is required to maximise resolution of
any chemical separation the phase achieves. UHPLC column technology
(sub-2μm and coreshell) decreases the diffusion distance of peptides
in-and-out of the porous media, resulting in sharper peaks, which
increases resolution, peak capacity and sensitivity of any peptide
mapping method.
Intact Protein Analysis by UHPLC
While one might assume that protein separations are similar to peptides
because of their similar amino acid composition, separation rules are
actually substantially different. While peptides are partitioned based
on their amino acid sequence, protein selectivity is significantly
influenced by the three-dimensional structure of a protein. As proteins
get larger, size and shape play a significant role in the retention of a
protein. Large proteins diffuse much more slowly than peptides; to
maximise protein separation performance, a much shorter diffusion path
is required. For fully porous silica media this requires very small
particles (sub 2μm) and for core shell media this requires a much
thinner porous shell than peptidespecific columns. In addition, mean
pore size for protein columns must be larger (approximately 250Ĺ plus)
to allow accessibility to the larger protein and out of the porous
layer. The lower surface area of such media results in a less
hydrophobic stationary phase, which is beneficial for intact protein
separations where recovery is always a major concern. An example of the
improved performance that a next generation UHPLC core shell column can
demonstrate is shown in Figure 3, where an IgG monoclonal antibody was
loaded on either a fully porous 5μm 300Ĺ C18 column or a core shell
3.6μm C18 column. Note the greater efficiency, recovery, and resolution
for the core shell column.
Intact protein analysis of large proteins can provide useful information
about a protein therapeutic and is complementary to peptide mapping
applications in understanding the purity and chemical stability of a
protein therapeutic. While peptide mapping is most useful for
determining site-specific modifications at a certain amino acid, peptide
mapping struggles to provide information about the global
threedimensional structure. Intact UHPLC analysis, on the other hand,
can provide some of this information as protein folding and global
structure both influence retention characteristics, thus intact protein
analysis can elucidate global structural changes that peptide mapping
misses. N-Terminal and C-terminal truncations and/or readthroughs can
often be identified and quantitated better by intact protein reversed
phase HPLC analysis. Indeed, the regulatory requirements for most
protein therapeutics demand intact reversed phase and gel filtration
analysis (for aggregate quantitation) as well as peptide mapping in most
regulatory filings. Next generation UHPLC columns like core shell
deliver more accurate, sensitive, and higher throughput solutions for
protein characterisation that are application specific and
characteristically different from peptide UHPLC columns.
Conclusion
As protein therapeutics have become larger and more complex, existing
analytical analysis solutions have proven inadequate to fulfil the
characterisation requirements of regulatory bodies. Fortunately,
analytical instrumentation and separation devices have realised
significant technological advances that meet current challenges. In
addition, performance advances in chromatography have reached a level
where using a 'one size fits all' approach can no longer provide the
separation requirements for all separations. Protein-specific and
peptide-specific UHPLC columns have been developed by multiple vendors
to meet the contrasting requirements of each applicationj and address
future analytical needs.
References
1. Mellors JS and Jorgenson JW, Anal Chem 76(18): pp5,441-5,450, 2004
2. van Deemter JJ, Zuiderweg FJ and Klinkenberg, A Chem Eng Sc 5: pp271-289, 1956
3. Gadgil H, Pipes G, Dillon T, Treuheit M and Bondarenko P, JASMS 17(6): pp867-872, 2006
4. Gritti F, Leonardis I, Shock D, Stevenson P, Shalliker A and Guiochon GJ, Chromatogr A 1217(10): pp1,589-1,603, 2010
5. McGinley M, Jarrett D, Layne J, Chitty M and Farkas T, Chromatography Today, pp33-36, November 2011
6. Meirovitch H, Radcousky S and Scheraga HA, Macromolecules 13: pp1,398-1,405, 1980
7. Davies PA, J Chromatogr 483: pp221-237, 1989
Acknowledgment
The author would like to thank Jeff Layne and Tivadar Farkas for their input into this article. |
Read full article from PDF >>
|
|
 |
|
| Rate this article |
You must be a member of the site to make a vote. |
|
Average rating: |
0 |
| | | | | |
|
|
Michael McGinley is Senior Product Manager at Phenomenex, Inc, where he is responsible for technical aspects of Phenomenex's HPLC and bioseparation product portfolios. Prior to joining Phenomenex in 2002, Michael was at Amgen where he held several positions over a 12-year period, including Associate Scientist, Project Manager and Laboratory Head. Michael received his BSc in Biochemistry from the University of California at Berkeley. His expertise includes HPLC method development, proteomics, biofuel analysis, oligonucleotide chemistry, protein characterisation and protein purification, and he is the author of more than 100 publications in these fields.
|
|
|
|
|
|
 |
News and Press Releases |
 |
Immutep Announces GMP Manufacturing Process Developed for IMP761, a First-in-Class LAG-3 Agonist for Autoimmune Disease
SYDNEY, AUSTRALIA – 06 December 2022 – Immutep Limited (ASX: IMM;
NASDAQ: IMMP) ("Immutep” or “the Company”), a clinical-stage
biotechnology company developing novel LAG-3 immunotherapies for cancer
and autoimmune disease, announced today that a GMP compliant
manufacturing process has been established for IMP761, its proprietary
preclinical candidate for autoimmune diseases. The 200L scale attained
by Northway Biotech, an end-to-end biopharmaceutical contract
development and manufacturing organisation (CDMO), will ensure supply of
IMP761 for IND-enabling studies and ensuing clinical trials.
More info >> |
|
 |
White Papers |
 |
|
|
