Developments in On-Line Chromatographic Methods Coupled with Mass Spectrometry for the Characterization of Intact Proteins

Paul Rainville, Himanshu Gadgil, Da Ren and Jeff Mazzeo

Waters Corporation, 34 Maple Street, Milford, MA 01757

INTRODUCTION Size Exclusion Chromatography (SEC) Affinity Chromatography Reversed-Phase Chromatography Ion-Exchange Chromatography (IEC)
LC-MS has become a powerful tool used in the characterization of 0.8mL/min
UV Step 1
complex samples. By far the most common mode of chromatography Loading & Washing Waste Gradient mobile phase conditions:
Tg= 25-50% B/15 min
coupled with mass spectrometry has been reversed-phase. The coupling 50 mM
1mL/min
0.3 mL/min A: 10 mM ammonium formate pH 5.0
Biosuite 250 5µm HR TM
50 mM B: 50 % formic acid
of reversed-phase chromatography with mass spectrometry has been a ammonium
ammonium Prototype Affinity MS
formate
very effective tool in the characterization of protein pharmaceuticals. 0.2mL/min Formate 0.5 mL/min
The most prominent use of this technique has been peptide mapping, MS pH 7.0
Protein-Pak CM 8HR UV MS
where peptides obtained from proteolytic cleavage of the protein 0.2mL/min

Step 2
pharmaceutical are separated and identified. The advantage of this
5% formic acid Elution and Analysis Waste Figure 9: Schematic and experimental conditions for IEC coupled with mass
technique is the direct identification of peptides as well as modifications 50% acetonitrile spectrometry.
that might be present. Modifications such as oxidation, deamidation 0.3 mL/min
1% formic acid MS 1
and glycosylation can all be determined using this technique. Here we Figure 1: Schematic and experimental conditions for SEC coupled with mass Prototype Affinity
Figure 6: IgG1 and other proteins are typically stored in salt containing buffer. 100
UV Detection
7.99

1. Cytochrome C
show the use of other modes of chromatography coupled with mass spectrometry. 0.1 mL/min Salt suppress ionization of proteins during ESI-MS analysis. Salts also 2. Horse Heart Myoglobin
spectrometry for the separation, identification, and characterization of acetonitrile form adducts with proteins increasing the heterogeneity in the sample. 2 3
%

1,000,000 Formate buffer 8.85 3. Enolase

1 thyroglobulin(600,000)

intact proteins. Chromatographic separation modes based on size Phosphate buffer

4
2 apo-transfferin dimer (160,000)
3 apo-transfferin monomer (80,000) Infusion of 20 picomoles of IgG1 in 20 mM Tris, 0.2% formic acid 50% 10.37

100,000 4 carbonic anhydrase (30,000)

acetonitrile is shown. A
-0

MS Detection
exclusion, ion exchange, reversed-phase, and affinity were evaluated.
3 5 cytochrome C (12,000)
Figure 4: Schematic and experimental conditions for affinity chromatography
7.98
100

1 6 lucine enkephalin (554)

Log MW

Absorbance (280 nm)

6
Each of these chromatographic separation modes coupled with mass 10,000 2 5 Formate buffer coupled with mass spectrometry. Small
Sample: 20 picomole IgG in 0.5M Tris buffer %

molecule
spectrometry offers a different dimension in the characterization and 1,000 3
4
impurities
Column: Protype desalting cartride
Buffer A: 0.2% Formic acid
8.84

10.60

separation of intact proteins. 1

2 6
Buffer B: 0.2% Formic in Acetonitrile
Gradient: 6.5%/min
0
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
Time

5 Phosphate buffer Flow rate: 0.2ml/min

100 IgG1 MS: Waters Micromass QTOF II
5 7 9 11 13 15
Rentention time 70% ACN
Retention time
Loading Elution
METHODS and MATERIALS Figure 2: Comparison of traditional vs. MS compatible SEC buffers on a standard 100

protein separation.
A Protein Impurities 893.35 942.86
100
16955.00

System Components 848.78

BSA monomer B % 998.27

Waters® BioSuiteTM Intact Protein System 808.37

%
771.73 1060.57

Waters® 2796 Separations Module. β Lactoglobulin

IgG1 5% ACN
738.21

707.48
1131.25 1211.93
1305.14
16978.00

Waters® 2487 Dual Wavelength Absorbance Detector 0

1413.81
m/z 0
10000 20000
mass
30000

A
500 600 700 800 900 1000 1100 1200 1300 1400

Waters Mircomass® ZQTM Mass Detector Cytochrome C

Figure 7: TIC showing separation of salts and other small molecule impurities
Waters Micromass® Q-TOF micro™ from intact IgG1. The separation conditions are shown in upper right.
Figure 10A: Separation and detection of 10µg of protein mixture containing
Columns BSA dimer
cytochrome c, horse heart myoglobin and enolase by IEC coupled
Reversed-phase: BioSuite™ desalting cartridge, 2.1 x 10 mm with UV and MS detection.
SEC: BioSuiteTM 250, 5 µm HR SEC, 7.8 X 300 mm Deglycosylated ……SHSLSPG Figure 10B: Protein ion envelope and deconvoluted spectrum confirming
IgG

Affinity: Prototype affinity column, 4.6 x 50 mm BSA dimer

B C 128

……SHSLSPGK
identity of peak 2 as horse heart myoglobin.

IEC: Protein-Pak CM 8HR, 4.6 x 250 mm BSA monomer

BSA dimer
CONCLUSIONS
BSA monomer 127.5
Lysine
Variant
BSA monomer
Experimental Conditions • IEC, SEC, and affinity chromatography can be directly coupled to
HPLC: Refer to Figures and Legends β Lactoglobulin mass spectrometry with mobile phase modifications.
MS: Source = ESI(+) Capillary (kV) = 3.3
B β Lactoglobulin
Figure 5A: On-line affinity-MS of crude ascites containing IgG1 is shown
above a flow-through peak possible containing non IgG protein impurities
can be seen at the retention time of 4.50 min. A smaller peak for IgG can
• Different modes of chromatography can be successfully coupled
Cone (V) = 25 and 30 (IgG1) Cytochrome C Cytochrome C
with mass spectrometry for the identification and characterization
also be seen eluting with 1% formic acid.
Temperature (ºC) Source = 150 Desolvation = 425 of intact proteins.
Gas Flow (L/Hr) Cone = 50 Desolvation = 500 Figure 3A: Separation of a four component protein mixture by SEC-MS. Figure 5B: Deconvolution of the major peak (4.50 min) shows a molecular
Scan Mode weight of 65,973 which agrees well with the molecular weight of albumin. • Affinity and reversed-phase chromatography where shown to iso-
Figure 3B: Protein ion envelope and deconvoluted spectrum is shown for all proteins in Figure 8: MS spectrum of IgG1 obtained after on-line desalting is shown. The late proteins from impurities that could interfere with analysis by
the sample mixture, the addition of the MS enables for direct identification. Figure 5C: Deconvoluted spectra of deglycosylated IgG1. A peak for a lysine amount of sample used (20 picomoles) is identical to that from Figure 6. mass spectrometry.
variant in IgG1 can also be seen. A distinct enhancement in signal-to-noise ratio can be observed after
online desalting.

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