manufacturing of biologics and other
drugs, says Barry L. Karger, a professor
of chemistry and director of the Barnett
Institute at Northeastern University. He
points to the use of microfluidic chips to
replace traditional LC separations at the
front end of MS as one example. “With
microfluidic chips, you have good reproducibility from run to run and chip to
chip,” and the process is fairly simple and
convenient, Karger says. “The more automation you can have, and the less human
interpretation and operation, the better.”
Last fall, the Barnett Institute launched the Center
for Advanced Regulatory
Analysis (CARA), which
aims to help transfer analytical techniques from the
basic research stage to the
drug development arena.
One CARA project has involved a partnership with
Momenta Pharmaceuticals
to use CE/MS to look at intact glycoforms of proteins.
Other glycosylation analysis techniques typically involve
cleaving glycans from proteins,
yielding the different forms, “but
you still haven’t put the molecule
together,” Karger says. Using CE/MS to
locate the glycans at specific sites on the
protein gives another level of information,
he says.
John R. Engen, a chemistry professor at
Northeastern and a fello w at the Barnett
Institute, is using MS to tackle questions
about protein structure. He and his coworkers take advantage of hydrogen/deu-terium exchange reactions to illuminate
overall protein conformation and dynamics. By using MS to monitor the exchange
of protein backbone amide hydrogens
with deuterium in solution, they recently
studied how glycosylation of the antibody
immunoglobulin G might affect the way it
interacts with its receptors (Anal. Chem.
2009, 81, 2644). The approach uses only picomoles
of material. Engen and colleagues envision that, with
appropriate software, the
technique could be used as
a screening tool to evaluate
how protein modifications
affect activity. A collaboration between Engen and
Waters Corp. aims to make
DYNAMIC The rate at
which a protein exchanges
hydrogen for deuterium
can be used to evaluate
the conformation of
immunoglobulin G. The
structure shown is a
model derived from crystal
structures of the antibody’s
antigen-binding regions
(top) and its constant
region (bottom).
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ANOTHER TECHNIQUE that Karger, Barnett Institute research professor Shiaw-Lin (Billy) Wu, and colleagues are working
on is using MS with a combination of both
ETD and CID to identify disulfide bridges
in proteins (Anal. Chem. 2009, 81, 112).
Disulfide bridges, which link the thiol
groups of cysteine residues, play a critical
role in the folding and stability of proteins.
“Disulfide scrambling” typically means an
incorrectly folded, inactive molecule. Putting CID and ETD together in a combined
LC/MS system reduces the need to run
multiple experiments to analyze disulfides, Karger says. The method even iden-tifies the interactions within tissue plasminogen activator ( TPA), a clot-dissolving
enzyme that folds with 17 disulfide bonds.
Complicated proteins, such as TPA, still
require manual interpretation of the data,
the researchers say, but analysis of simpler ones—for example, human growth
hormone, which has only two disulfide
linkages—can be automated.
such instrumentation commonplace (Anal.
Chem. 2008, 80, 6815).
With all of the data that biologic drug
researchers have in hand, two important
issues remain outstanding: What key properties are scientists currently missing in
their analyses of biologics, and what is the
clinical significance of all of the analytical
data they currently do obtain?
The first question stems directly from
the heparin adulteration that rocked the
pharmaceutical industry last year (C&EN,
ANAL. CHEM.
