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INTRODUCTION
Residual host cell proteins (HCPs) are low level
contaminants in biotherapeutics that may elicit
an unpredictable immune response and need
to be monitored as part of regulatory
guidelines . The identification and quantitation
of ppm-level HCPs in biopharmaceuticals using
two-dimensional chromatography and data-
independent mass spectrometry has been
shown in previous work1,2. The incorporation of
ion mobility to resolve peptides in multiple
dimensions is shown here.
Improved Identification and Quantitation of Host Cell Proteins in Protein Therapeutics Using 2D-LC and Ion Mobility Mass Spectrometry
Martha Stapels, Catalin Doneanu, Keith Fadgen, Stephane Houel and Weibin Chen
Pharmaceutical Life Sciences, Waters Corporation, Milford, MA 01757
METHODS
LC/MS: Prototype nanoACQUITY UPLC®
System with 2D Technology
SYNAPT™ G2-S HDMS™
Sample Preparation: Proteins were
solubilized with Rapigest, reduced, alkylated,
and digested in-solution with trypsin.
First Dimension:
Column: 300 µm x 5 cm XBridge C18 (5 µm)
Gradient formation: discontinuous step
gradient
Eluent A: 20 mM ammonium formate pH 10.0
Eluent B: acetonitrile
Second Dimension:
Trap: 180 µm x 2 cm Symmetry C18 (5 µm)
Column: 75 µm x 15 cm HSS T3 C18 (1.8 µm)
Eluent A: 0.1% formic acid in water
Eluent B: 0.1% formic acid in acetonitrile
Online Dilution with RP/RP: To maximize
sample recovery on the second dimension trap
column from the organic-containing fractions,
an aqueous flow was delivered with the 2nd
dimension pump, and mixed with the eluted
fraction prior to trapping.
MS Data processing : Data were processed
and searched with ProteinLynx Global Server
(PLGS 3.0) with IdentityE informatics.
RESULTS
Figure 1. Fluidic layout for 2D RP/RP LC used in this study.
Figure 6. PPM levels of proteins identified and quantified with 1D
LC HDMSE, 2D 5-Fraction LC MSE, 2D 3-Fraction LC HDMSE, and 2D 5-Fraction LC HDMSE. Additional peak capacity via 2D LC or
ion mobility enables lower level analysis.
Orthogonal dimensions of fractionation facilitate
the identification and quantitation of HCPs down to the ppm level. MassPREP Digestion Standard Mix 2 (BSA, Enolase, PhosB, and ADH) and the Hi3 Ecoli Standard ClpB were spiked into a trastuzumab digest at
levels ranging from 5 to 514 ppm to test the performance of the system with different methods. As seen in Figures 6-8,
adding orthogonal steps with 2D fractionation or ion mobility
is necessary to identify and quantify peptides down to 5 ppm. This method was next applied to the Intact mAb Mass Check Standard, which is a mouse IgG purified by
Protein-A. Mix 2 and the Hi3 Ecoli Standard ClpB were
spiked into this sample as well, as shown in Figure 9. A total
of 73 proteins from mouse were identified in this sample, at levels ranging from 2 to 500 ppm, shown in Figures 10 and
11. The application of this technology to a biopharmaceutical sample in two different labs can be seen in Figure 12.
Figure 4. BPI chromatograms for a 5 fraction HCP analysis.
A total of 8.3 µg of trastuzumab digest was injected onto the 1st dimension. Total run time was 5 hours.
CONCLUSION
MSE and HDMSE provide unbiased HCP identification and quantitation
Increasing peak capacity with orthogonal methods is beneficial for identifying and quantifying ppm level HCPs
Ion mobility resolves interfering peptides and requires no additional analysis time
HDMSE removes interfering fragment ions to enable identification by standard proteomic software tools
REFERENCES
1. Schenauer, M.R. et. al. Analytical Biochemistry.
2012 (428):150-157. 2. Doneanu, C. et. al. mAbs. 2012 (4):24-44.
Figure 12. Identified HCPs with corresponding ppm levels in a
biotherapeutic sample by two different laboratories. SYNAPT G1 results generated by an unnamed biotech company.
Figure 10. Log PPM levels of proteins identified and quantified
with 1D LC MSE (blue), 1D LC HDMSE (red), and 2D 5-Fraction LC HDMSE (green) in a mouse IgG purified by Protein-A.
Spiked protein standards shown with diamonds. Level of the product protein shown to demonstrate total dynamic range of
the experiment.
Ion mobility
Figure 7. BSA peptide at 514 ppm and 51 ppm in 8.3 µg of
trastuzumab processed by TransOmics Informatics for Proteomics. Using only 1D LC and HDMSE, peptides could be
identified and quantified to 50 ppm.
Figure 2. Alignment of low energy and elevated energy ions by
LC retention time (top, MSE) and also with the orthogonal dimension of separation by mobility (bottom, HDMSE).
Figure 3. A plot showing the three resolving axes used in this
analysis: LC retention time, m/z, and ion mobility drift time. Each of these axes has a given resolving power (N) and the overall
system resolving power is given by the product of all three and the fraction of occupied bins. The incorporation of ion mobility
into this analysis increases the peak capacity of the system by at least an additional order of magnitude.
Figure 5. HDMSE spectra of a peptide from enolase at 112
ppm (top) and 11 ppm (bottom) in 8.3 µg Herceptin digest. Figure 11. PPM levels of proteins identified and quantified with
1D LC MSE (blue), 1D LC HDMSE (red), and 2D 5-Fraction LC HDMSE (green) in a mouse IgG purified by Protein-A. Spiked
protein standards shown with diamonds.
Figure 9. Spiked vs. measured ppm levels of 5 standard
proteins in a mouse IgG purified by Protein-A. Protein concentrations were determined down to 10 ppm with an
average of 9% RSD.
Figure 13. Mascot identification of a HCP peptide identified at
10 ppm in a biotherapeutic sample. HDMSE removes interfering fragment ions to enable validation by standard
proteomic software tools.
0
50
100
150
200
250
300
350
0 100 200 300 400 500 600
Spik
ed
PP
M
Measured PPM
Without Ion Mobility
With Ion Mobility
514 ppm 51 ppm
Measured Ratio:9.08
MS A B C D E F G H I J K Sum Ave CV
SYNAPT G1 MS 20 46 4 70 30%
SYNAPT G2-S MS 42 9 6 43 11 7 2 120 22%
SYNAPT G2-S HDMS 32 12 7 71 10 4 3 8 8 4 3 162 12%
50+ ppm
25+ ppm
10+ ppm
1+ ppm
Figure 8. BSA peptide ranging from 514 ppm to 5 ppm in 8.3 µg
of trastuzumab processed by TransOmics Informatics for Proteomics. Using 5-Fraction 2D LC and HDMSE, peptides could
be identified and quantified down to 5 ppm.
514 ppm 51 ppm 5 ppm
% (
max =
28753)
% (
max =
2164)
log p
pm