Reversed-Phase HPLC Method for Hydrophobic Protein Analysis
Jasmine Wang, Bhargavi Vemulapalli, Susanna Bilbulian, Liu Tie, Huiming Yan, Ziping Wei, and Patricia W. CashAnalytical Biochemistry Department, MedImmune, Gaithersburg, MD. USA
Abstract

Protein Quantification: Linearity

Protein Identity Method: Peak Identification by LC/MS

Protein Identification: Gradient Optimization

13.18

A reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed
to measure the concentration of intact hydrophobic protein and the level of protein fragments. It isvery difficult to elute the hydrophobic protein from traditional reversed-phase columns. The method
described here employs an Intrada WP-RP column packed with a newly developed reversed-phaseligand. The ligand has an optimal surface polarity that increases column efficiency duringhydrophobic protein analysis (1). The elution of the protein and separation of fragments wasachieved by optimizing the gradient.
The protein concentration in the test sample was determined using regression analysis from astandard curve. The determinations were performed in the linear range of 1 – 10 µg. The method
qualification results demonstrate that the method is specific, precise, accurate, and linear. Themethod is compatible with on-line ESI LC/MS detection for the identification of the protein - relatedimpurities.

Area

57.5 µg5 µg02.5 µg

Correlation: 1.00000
Amount[µg]

50

2

4

4.00

1 µg

68

1012

141624

Hydrophobic Protein

tedr rela
BuffemagFr System: Agilent 1100/1200 HPLC Analytical Column: Intrada WP-RP (4.6 x 250 mm, 3 μm 300Å) Column Temperature: 35°C
 Mobile Phase:A: 0.1% TFA/HPLC Water
B: 0.1% TFA/Acetonitrile
 Flow Rate: 0.75 ml/min
 Autosampler Temperature: 4°C
 Sample Load: 10 µg
 UV Detection: 280 nm
 Gradient: 0-100% B in 18 minutes

300

600

900

1416Gradient B
With slope at 6for % B increasing

tsen

1
Day
2

1Analyst
21Figure 1. Recovery of Hydrophobic Protein in Cell Culture Medium
Purified protein was spiked into the medium and analyzed following the above procedure. The spikerecovery was determined to be 91% of the theoretical value.

Instrument
2Fragment 14.445.01

12.00

14.00

16.00

18.00

Intact Protein

B: RP-UPLC profile ofhydrophobic protein heatstressed, 40°C,
3 months

BFragment 3

6.14
5.96
10.27

Fragment 4Resolution = 1.4
Time
4.00

6.00

8.00

02

4

68

10

12

14

16

10.00

12.00

14.00

16.00

18.00

18 minProtein Identification: Repeatability

Figure 4. Gradient Optimization
Gradient elution played a key role in the separation of the fragments. The optimal resolution was achieved with gradient B.

Reported Fragmentation

Protein Identification: Specificity

A% CV

Test sample

Reported Intact Protein
Std Dev

Total PeakArea, %A: RP profile of heat-stressedblank buffer

Buffer related% CV

Mean PeakArea, %

1.8
02468101214160.5182
0.5628Mean PeakArea, %

99.5779

0.4040Hydrophobic
Protein

3.8

Std Dev % CV
Total PeakArea, %

0.4221

3.5

99.5960
0.5

0.1

13.3

99.4818
99.43720.430199.5699

0.496899.5032

99.5

0.1

0.1

18 minTable 5. Results of Repeatability Evaluation
Hydrophobic
ProteinBProtein Quantification: Intermediate Precision

Hydrophobic
Protein

10.00

Figure 6. Specificity Evaluation

The accuracy of the method was evaluated by analyzing the test sample in triplicates at three different levels. The % CV
between the three replicates at each level was 1.8-3.8.

Hydrophobic Protein

8.00

Fragment 2

Table 2. Results of Accuracy Evaluation

Protein Quantification: Specificity

6.00

18min

The fragment peaks and clipping sites were identified by Waters LCT mass spectrometry.

Protein Quantification: Accuracy
Measured Concentration (µg/ml)
Value
Mean
290.3
300
298.4
311.1
576.6
588
573.9
613.8
878.4
875.7
886
904.7

121.4

The repeatability of the method was evaluated by analyzing the test sample in six replicates. The % CV was 1.4.

Hydrophobic Protein(µg/ml)

10% CV

Table 1. Results of Repeatability Evaluation

Experimental Parameters

8Hydrophobic Protein

B

Measured Concentration (µg/ml)
Value
Mean
926.4
925.7
919.9
914.0
912.6
905.8
893.8

618 minProtein Quantification: Repeatability
Test Sample

A: RP-UPLC profile ofhydrophobic protein

12.130The UV responsed are linear in the range of 1-10 µg. Correlation coefficient was 1.0.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is one of the most usefultechniques for the separation of proteins. In this technique, proteins are separated based on theirhydrophobic properties. The retention of proteins with high hydrophobicity is a major concern in thistype of protein analysis by HPLC (2, 3).
Here we present a reversed-phase chromatographic method that implements an Intrada WP-RP
column packed with a high resolution silica matrix with 3µm particles and 300Å pore size. Themethod allows us to separate, identify, and quantitate a hydrophobic protein in cell culture medium
and in-process samples.

A4.46 4.62

Fragm3
2
1

Resolution = 1.2

ents4

Intact Protein

Gradient A
With slope at 7
for % B increasing

tedr rela
BuffeFigure 2. Linearity Evaluation

Introduction

Hydrophobic Protein

A10 µgMeasured Concentration (µg/ml)
Value
Mean
520.4
523.7
527.0
519.0
537.0
527.0
526.2
518.3
537.0
531.6
526.2
518.3
580.4
566.8
580.6
539.5
513.7
515.6
517.4
513.8
520.4
522.1
527.0
519.0

mag
Fr% CV

B: RP profile of testsample

tsenConclusion• An Intrada WP-RP column was implemented for a protein with high hydrophobicity.
• The method allowed us to quantitate the protein concentration and separate the protein related fragments.
• Separation of protein fragments was successfully achieved with the optimized gradient.
• The fragments were identified in heat stressed samples by a RP-UPLC system coupled to a Waters LCT
mass spectrometer.
• The qualification results demonstrated that the method was specific, precise, accurate, and linear.

0.5024681012141618 minHydrophobicProtein

C
4.5

C: RP profile of heatstressed sample

Buffer relatedmag
Fr

The repeatability of the method was evaluated by analyzing the test sample in six replicates. The % total fragment
peak was determined to be 0.5 and % CV was 13.3.

References
1. Imtakt Intrada WP-RP HPLC Column Brochure
http://www.sisweb.com/lc/imtakt-intrada-wprp.htm
2. C. Guerin, G. Brule, Separation of three proteins from egg white, Sciences des Aliments, 12(4), 705-20(1992)
3. A. Schafer, the Quality of Egg and eggproducts, Fourth Eur. Symp (1995)

tsen

0.9

Acknowledgement024681012141618 min

Table 3. Results of Intermediate Precision Evaluation

Figure 5. Specificity Evaluation

The intermediate precision was evaluated by different analysts, on different days and on different instruments .
The % CV was found to be 0.5 – 4.5.

No interfering peaks were detected in the blank buffer. Additional peaks were observed in the heat-stressed sample.
The assay is specific.

35th International Symposium on High Performance Liquid Phase Separations and Relation Techniques – HPLC 2010, June 19-24, 2010, Boston, MA USA. P-322-M

The authors would like to acknowledge Dr. Wenjun Mo, Dr. Mark Schenerman, and Dr. Gail Folena Wasserman for their support of this work.