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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