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Using Temperature to Improve Peak Shape of Hydrophobic Proteins in Reversed-Phase HPLC

Roy Eksteen, Dave Bell, and Hillel Brandes Supelco, Div. of Sigma-Aldrich Bellefonte, PA 16823 USA

T414073


Background

Temperature has been used in reversed-phase HPLC (RPC) to improve peak shape and efficiency for small MW compounds and has also been found generally beneficial in terms of reducing analysis time, albeit at the general cost of lowering selectivity. With proteins, peak shape in RPC is generally enhanced by parameters that stabilize a single denatured state (1). Temperature is one way that can dramatically affect the tertiary and quaternary structure of proteins, and thus a “denatured state”. As such, we investigated the impact of column temperature (up to 90 ºC) on RPC of several globular proteins and monoclonal antibodies (mAbs). While retention decreases with increasing temperature, peak shape of most proteins was dramatically affected by temperature. However, when operated under optimal conditions, peak shape and resolution of antibodies on several commercial columns was superior on a BIOshell™, wide pore Fused-Core® column. In general, under optimal conditions, the wide pore Fused-Core column exhibited narrower peak widths, higher peak heights, better resolution, and thus greater sensitivity.

2


Experimental Monoclonal antibodies were made available as purified concentrated stocks in PBS, by Kevin Ray, Sigma-Aldrich, St. Louis. Other globular proteins to serve as controls, were procured from Sigma-Aldrich: albumin (BSA), A7906; carbonic anhydrase (CA), C3934; ribonuclease (RNAse), R5500. Columns: BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 µm Aeris WIDEPORE C4, 10 cm x 2.0 mm I.D., 3.6 µm XBridge BEH300 C4, 10 cm x 2.1 mm I.D., 3.5 µm Zorbax SB300 C3, 10 cm x 2.1 mm I.D., 3.5 µm Chromatographic Conditions: mobile phase A: 80:20, (water, 0.1% TFA) : (acetonitrile, 0.1% TFA) mobile phase B: 50:50, (water, 0.1% TFA) : (acetonitrile, 0.1% TFA) flow rate: 0.4 mL/min gradient 1*: 40 to 100%B in 10 min (1.8% acetonitrile/min; 6%B/min) gradient 2*: 0 to 96%B in 16 min (1.8% acetonitrile/min; 6%B/min) column temp.: as indicated det.: UV, 215 nm injection: 0.2 µL, 1 g/L mAb in 20 mM Tricine/KOH, pH 7, or 0.5 µL, 1 g/L ea control proteins in 20 mM Tricine/KOH

3 *gradient 1 for mAbs; gradient 2 for other proteins


Experimental (contd.)

Column Pore (Å) dp (µm) Type #Max.

Temp. ºC BIOshell A400 Protein C4 400 3.4 core-shell 90 Aeris® WIDEPORE C4 200 3.6 core-shell 60 XBridge® BEH300** C4 300 3.5 porous 80 Zorbax® SB300 C3 300 3.5 porous 80

** a.k.a. PrST # Maximum temperature recommended by vendor

4


Figure 1. Reversed-Phase mAb Elution

5

SIGMAMab

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

Temperature, oC

Peak

Are

a


Erbitux

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

6

Figure 1. Reversed-Phase mAb Elution (contd.) Pe

ak A

rea

Temperature, oC


Humira

0.0

1.0

2.0

3.0

4.0

5.0

6.0

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

7


ak A

rea

Temperature, oC


Peak

Hei

ght,

mAU

0

10

20

30

40

50

60

70

80

90

20 40 60 80 100

8

Figure 1. Reversed-Phase mAb Elution (contd.)

SIGMAMab

Temperature, oC


0

10

20

30

40

50

60

20 40 60 80 100

9


ak H

eigh

t, m

AU

Erbitux

Temperature, oC


0

20

40

60

80

100

120

20 40 60 80 100

10


ak H

eigh

t, m

AU

Humira

Temperature, oC


Peak

Wid

th (1

/2 h

eigh

t), µ

L

0

10

20

30

40

50

60

70

20 40 60 80 100

11

Figure 1. Reversed-Phase mAb Elution (contd.)

SIGMAMab

Temperature, oC


0

10

20

30

40

50

60

20 40 60 80 100

12


ak W

idth

(1/2

hei

ght),

µL

Erbitux

Temperature, oC


0

10

20

30

40

50

60

20 40 60 80 100

13


ak W

idth

(1/2

hei

ght),

µL

Humira

Temperature, oC


Fig 2. Reversed-Phase Elution of Control Proteins

RNAse

Peak

Are

a

6.5

7.0

7.5

8.0

8.5

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

14

Temperature, oC


BSA

9.0

9.5

10.0

10.5

11.0

11.5

12.0

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

15

Fig 2. Reversed-Phase Elution of Control Proteins (contd.) Pe

ak A

rea

Temperature, oC


Carbonic Anhydrase

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

20 40 60 80 100

BIOshell

Aeris

XBridge

Zorbax

16


ak A

rea

Temperature, oC


Peak

Hei

ght,

mAU

75

85

95

105

115

125

135

145

155

165

20 40 60 80 100

17

Fig 2. Reversed-Phase Elution of Control Proteins (contd.)

RNAse

Temperature, oC


30

35

40

45

50

55

60

65

70

75

80

20 40 60 80 100

18


ak H

eigh

t, m

AU

BSA

Temperature, oC


60

70

80

90

100

110

120

130

140

150

20 40 60 80 100

19


ak H

eigh

t, m

AU

Carbonic Anhydrase

Temperature, oC


Peak

Wid

th (1

/2 h

eigh

t), µ

L

15

20

25

30

35

40

20 40 60 80 100

20

Fig 2. Reversed-Phase Elution of Control Proteins (contd.)

RNAse

Temperature, oC


20

30

40

50

60

70

80

90

100

110

120

20 40 60 80 100

21


ak W

idth

(1/2

hei

ght),

µL

BSA

Temperature, oC


20

25

30

35

40

45

50

55

20 40 60 80 100

22


ak W

idth

(1/2

hei

ght),

µL

Carbonic Anhydrase

Temperature, oC


Fig 3. Example of Comparative Optimal Elution of SIGMAMab

-5

5

15

25

35

45

55

65

75

2.7 2.9 3.1 3.3 3.5 3.7-5

5

15

25

35

45

55

65

75

3.3 3.5 3.7 3.9 4.1 4.3

-5

5

15

25

35

45

55

65

75

2.3 2.5 2.7 2.9 3.1 3.3-5

5

15

25

35

45

55

65

75

3 3.2 3.4 3.6 3.8 4

Elution Time, min

mAU

, 21

5 nm

BIOshell 75 ºC

Aeris 60 ºC

Zorbax 75 ºC

XBridge 75 ºC

23


Column QC: Check on Column Efficiency Plate Count for Toluene, (k = 2); n = 3

Column Initial Final Percent BIOshell 12,688 12,200 96% Aeris 13,487 11,501 85% XBridge 12,737 11,558 91% Zorbax 10,239 9,635 94%

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Conclusions • As expected, higher temperature consistently reduces elution time of all proteins

studied. • Monoclonal Antibodies

– Efficient elution of the mAbs from short-chain aliphatic reversed-phase columns requires elevated temperature.

– Optimal column temperature for the reversed-phase chromatography of monoclonal antibodies is in the range of 60 – 75 ºC.

– Optimal temperature results in narrower peak widths and greater peak heights for mAbs, while generally not adversely affecting recovery, as assessed by peak area.

• Globular proteins – Globular proteins eluted efficiently at moderate temperature. – For the control proteins, declining peak area at high temperature may reflect

protein degradation. Further studies are needed to clarify our results. – The reduction in peak area of proteins at highest temperature, most clearly for

BIOshell, can at least partially be attributed to resolution of contaminants from the main peak.

• The wide pore (400 Å), Fused-Core BIOshell column consistently outperformed the other columns examined, when comparing optimal conditions. 25


References

1. Nugent, K.D. et.al. Separation of Proteins by Reversed-Phase High-Performance Liquid Chromatography II. Optimizing Sample Pretreatment and Mobile Phase Conditions. J Chromatogr., 1988, 443, 381-397.

2. Fakete, S. et.al. Impact of mobile phase temperature on recovery and stability of monoclonal antibodies using recent reversed-phase stationary phases, J. Sep Sci., 2012, 35, 3113-3123.

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BIOshell is a trademark of Sigma-Aldrich Co LLC. Fused-Core is a registered trademark of Advanced Materials Technology, Inc. Zorbax is a registered trademark of Agilent Technologies, Inc. XBridge is a registered trademark of Waters Corporation. Aeris is a registered trademark of Phenomenex.

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