Optimized Superficially Porous Particles for Peptide and Protein Analysis

Joseph J. Kirkland, Stephanie A. Schuster, Brian M. Wagner, Barry E. Boyes, and

Joseph J. DeStefano

Advanced Materials Technology, Inc., 3521 Silverside Rd., Wilmington, DE 19810

For several years we have been designing and producing superficially porous (Fused-core®) particles for HPLC columns. The characteristics of these particles were specifically created to separate certain solutes optimally, usually based on molecular size. The original 2.7 µm superficially porous particles were created with an average pore size of 90 Å, which was suitable for small molecule analytical separations. This particle technology has been expanded to include wider pore sizes and larger particle sizes that are specifically designed for larger biomolecules. Novel particle designs with specially selected bonded phases for peptide and protein separations are described. This presentation includes fast separations of peptides and intact protein mixtures, as well as examples of very high resolution separations of larger proteins and associated variants and contaminants. Columns with bonded phases for these particles demonstrate high temperature stability, which is ideally suited for the conditions that are often used for analytical and small scale preparative biomolecular separations. Protein recovery and sample loading investigations are included. The optimized shell thickness of the new particles represents a compromise between a short diffusion path versus adequate retention and mass load tolerance. Examples of high molecular weight protein separations highlight the advantages of using columns of superficially porous particles with wider pores. Some comparisons with conventional totally porous particles are also shown.

Abstract

Fused-Core

Particle

Particle

Size

(µm)

Pore

Size

(Å)

BET

Surface

Area

(m2/g)

Shell

Thickness

(μm)

%

Porosity

Pore

Volume

(cm3/g)

Separation

Utility

Halo

2.7

90

135

0.5

75

0.26

Small molecules

< 5000 MW

HALO-5 4.6 90 90 0.6 56 0.21 Small

molecules < 5000 MW

Halo Peptide

2.7

160

80

0.5

75

0.29

Peptides

< 15 kDa

HALO-5 Peptide 4.6 160 60 0.6 56 0.21 Peptides

< 15 kDa

HALO Protein

3.4

400

15

0.2

31

0.11

Proteins

< 400 kDa

Physical Characteristics of Fused-Core Particles

Fused-Core

Particle

Particle Size

(µm)

Pore Size

(Å)

BET

Surface Area (m2/g)

Shell

Thickness (μm)

Separation Utility

Halo

2.7

90

135

0.5

Small molecules

< 5000 MW

HALO-5 4.6 90 90 0.6 Small

molecules < 5000 MW

Halo Peptide

2.7

160

80

0.5

Peptides

< 15 kDa

HALO-5 Peptide 4.6 160 60 0.6 Peptides

< 15 kDa

HALO Protein

3.4

400

15

0.2 Proteins

< 400 kDa

Physical Characteristics of Fused-Core Particles

Sample Loading Study for Halo 400 C4Columns: 4.6 x 100 mm; Gradient: 39 - 49% in 10 min.;

Mobile phase: A - 0.1% aqueous trifluoroacetic acid;B - acetonitrile with 0.1% trifluoroacetic acid; Temperature: 60 oC;

Flow rate: 0.5 mL/min; Injection volume: 5 mL

mg Apomyoglobin

0.01 0.1 1

Peak

wid

th, m

in.

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Shell thickness, 0.15 mmShell thickness, 0.20 mmShell thickness, 0.25 mm

Apomyoglobin – 17 kDa

The particle with 0.20 µm shell thickness offers a compromise between sample loadability and retention, as well as an optimized diffusion path for large MW biomolecules.

Sample Loading Study for HALO Protein C4

Columns: 4.6 x 100 mm; Gradient: 39-49% B in 10 min. Mobile phase: A – 0.1% trifluoroacetic acid

B – acetonitrile with 0.1% trifluoroacetic acid; Temperature: 60 oC Flow rate: 0.5 mL/min.; Injection volume: 5 µL

0.2 µm

3.0 µm 3.4 µm

Shell with 400 Å pores

Solid Core

HALO Protein

HALO-5 Peptide

0.6 µm

3.3 µm 4.6 µm

Shell with 160 Å pores

Solid Core

HALO® Wide-Pore Fused-Core Particles

SEM of Images of HALO Protein Particles

FIB (Focused Ion Beam) sliced particle

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Comparison of Bonded Phases Columns: 2.1 x 100 mm HALO Protein Instrument: Shimadzu Nexera Injection Volume: 1 µL Detection: PDA @ 280 nm Temperature: 60 oC

Flow rate: 0.5 mL/min Mobile Phase A: water/0.1% TFA Mobile Phase B: 80/20 ACN/water/0.1% TFA Gradient: 35-66.3% B in 5 min.

1

2

3

4

5 6 7

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

C4 3.4 µm 400 Å

ES-C8 3.4 µm 400 Å

ES-C18 3.4 µm 400 Å

Time, min.

1

2

3

4

5,6

7

1

2

3 4

5 6 7

Sample: In order 1. Cytochrome c 12.4 kDa 2. Lysozyme 14.3 kDa 3. α-chymotrypsin 25 kDa 4. Catalase 250 kDa (4 x 60 kDa) 5. Enolase 46.7 kDa 6. Carbonic anhydrase 29 kDa 7. β-amylase 200 kDa (4 x 50 kDa)

0

1

2

3

4

5

6

7

8

9

0 5000 10000 15000

Ret

entio

n Ti

me

(min

.)

Column Volumes

Halo Protein C4 @ 90 oC

cytochrome c Lysozyme apomyoglobin

catalase enolase

Column Stability Study

Column: 2.1 x 100 mm; Mobile phase gradient: 25-40% acetonitrile/0.1% aqueous trifluoroacetic acid in 10 min; Temperature: 90 oC; Flow rate: 0.5 mL/min; Detection: 215 nm

• The HALO Protein C4 bonded phase is stable up to 90 oC, showing very little loss of retention.

Column: 2.1 x 100 mm HALO Protein C4 Instrument: Agilent 1200 SL Injection Volume: 2 µL Detection: 215 nm Temperature: as indicated

Mobile Phase A: water/0.1% TFA Mobile Phase B: acetonitrile/0.1% TFA Gradient: 28-58% B in 10 min. Flow rate: 0.45 mL/min

Peak Identities (in order): 1. Lysozyme 14.3 kDa 2. BSA 66.4 kDa 3. α-Chymotrypsinogen A 25.0 kDa 4. Enolase 46.7 kDa 5. Ovalbumin 44.0 kDa

0 1 2 3 4 5 6 7 8 9 10

Time, min

30 oC

60 oC

90 oC

1

2

3

4

5

1

2 3 4 5

1 2 3

4 5

• Protein peak shape and recovery improve with increased temperature of analysis.

Protein Separations: Effect of Temperature

-20

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Abso

rban

ce, m

AU

Time, min

-20

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30 35 40 45

Abso

rban

ce, m

AU

Time, min

Totally porous, 300 Å, C4,1.7 µm P = 134 bar Flow rate = 0.2 mL/min Gradient = 20-60% B in 45 min. A = water/0.1% TFA B = Acetonitrile/0.1% TFA Temp = 60 oC

• Separation is 3 times faster at the same back pressure on the HALO Protein column compared to the same sample run on a sub-2-µm totally porous particle column

Peak Identities: 1. Ribonuclease A 2. Cytochrome c 3. BSA 4. Apomyoglobin 5. Enolase 6. Phosphorylase b

1

2 3

4

5

6

1

2 3

4

5

6

HALO Protein, 400 Å, C4, 3.4 µm P = 132 bar Flow rate = 0.6 mL/min Gradient = 20-60% B in 15 min. A = water/0.1% TFA B = Acetonitrile/0.1% TFA Temp = 60 oC

Protein Separations: Fused-Core compared to Totally Porous

• Proteins were fraction collected from a 4.6 x 100 mm HALO Protein C4 column run at 60 oC under gradient conditions with water/ACN/0.1% TFA mobile phase. Blanks were obtained by replacing the column with a union

• Lyophilized proteins were reconstituted using 3 M Urea/1% Triton X-100/0.25% acetic acid

• Protein recoveries were measured using QuantiPro™ BCA Assay Kit for 0.5-30 μg/mL protein (Sigma-Aldrich, St. Louis, MO)

• Samples were incubated at 37 oC for 100 min. • Each sample was run in duplicate • Absorbance values were measured at 562 nm • HALO Protein C4 shows good recovery of proteins

Protein Recovery Cytochrome c 100 (5.8 SD)

Catalase 92 (18 SD)

Protein Recovery Studies

Columns: 2.1 x 100 mm Instrument: Shimadzu Nexera Injection Volume: 1 µL Detection: 280 nm Temperature: 60 oC

Mobile Phase A: water/0.1% TFA Mobile Phase B: 80/20 ACN/water/0.1% TFA Gradient: 40-47% ACN in 10 min. Flow rate: 0.3 mL/min

Peak Identities (in order): 1. Catalase 250 kDa [~60 kDa subunit] 2. α-Chymotrypsinogen A 25.0 kDa 3. β-Galactosidase 465 kDa [116 kDa subunit] 4. β-Amylase 200 kDa [~50 kDa subunit]

Time, min 0 1 2 3 4 5 6 7 8 9 10

HALO Protein, C4 400 Å

Competitor SPP, C4 200 Å

0.0908

0.0767 0.0722 0.0614

0.0940

0.0699

0.2093

0.0912

Protein Separations: Effect of Pore Size

• Peak widths in minutes provided above each peak. • The 400 Å pores of HALO Protein enable sharp peaks for high MW biomolecules.

-5

45

95

145

195

245

295

0 5 10 15 20 25 30 35 40 45

Abso

rban

ce @

215

nm

Time, min

Column: 4.6 x 100 mm, HALO-5 Peptide ES-C18 Instrument: Agilent 1100 Injection Volume: 10 µL Detection: 215 nm Temperature: 45 oC Pressure: 54 bar initial

Mobile Phase A: water/0.1% TFA Mobile Phase B: ACN/0.1% TFA Gradient: 5-40% B in 45 min. Flow rate: 1.0 mL/min Sample: Apomyoglobin Tryptic Digest [2 mg/mL]

The extremely low back pressure of the HALO-5 Peptide ES-C18 column enables fast, efficient proteomic separations with a low potential for plugging.

Tryptic Digest using HALO-5 Peptide ES-C18

reduction

alkylation +

Column: 2.1 x 100 mm HALO Protein C4 Instrument: Shimadzu Nexera Injection Volume: 1 µL Detection: 280 nm Temperature: 80 oC

Mobile Phase A: water/0.1% TFA Mobile Phase B: 80/20 ACN/water/0.1% TFA Gradient: 33-40% B in 10 min. Flow rate: 0.25 mL/min

Sample: 0.5 mg/mL IgG1 treated with 100 mM DTT in 8 M Guanidine HCl at 50 oC for 35 min. Sample reduced only.

denaturation

2 Light Chains + variants

2 Heavy Chains + variants

Antibody structure: Afaneh, C, Aull, MJ, Kapur, S, Modern Immunosuppression Regimens in Kidney Transplantation, 2012.

-5000

0

5000

10000

15000

20000

25000

0 1 2 3 4 5 6 7 8 9 10

Abso

rban

ce @

280

nm

Time (min.)

-500

500

1500

2500

5 5.5 6 6.5

Abs

@ 2

80 n

m

Time (min.)

Separation of Reduced IgG1 using TFA Mobile Phase

ID Mass (Da)

LC1 23,204

LC2 23,192

LC3 23,203

HC1 50,539

HC2 50,424

HC3 50,668

HC4 50,680

HC5 28,862

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

LC1

LC2

LC3

HC

1

HC

2 H

C3

HC

4

HC

5

2.1 mm ID x 100 mm HALO Protein C4; 0.4 mL/min.; A: 0.5 % formic acid with 20 mM Ammonium Formate B: 45% AcN/45% IPA/ 0.5 % formic acid with 20 mM Ammonium Formate; Gradient: 29-32% B in 20 min.; 80 oC

Detection: 280 nm Abs; Shimadzu LCMS-2020, ESI +4.5 kV, 2 pps, 500-2000 m/z

500 750 1000 1250 1500 1750 m/z 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

Inten. (x100,000)

1366.0

1290.0

1221.9

1161.4

1105.9 1451.6

1655.6 1546.7

1010.1 1783.1

1931.5 930.2

859.4 1604.6 773.6 1859.1

LC = light chain HC = heavy chain

Masses deconvoluted

using MagTran

Abs

orba

nce

@ 2

80 n

m

High Resolution Analysis of mAb IgG1 Light and Heavy Chains with LC/MS

• Fused-core particles with 400 Å pores are effective for efficiently

separating proteins without restricted diffusion • Protein separations can be run approximately 3 times faster on columns

of Fused-core particles compared to columns of sub-2-µm particles at the same back pressure

• Fused-core particles have performance advantages over totally porous particles for separating peptides and proteins

• Columns of 400 Å particles are both efficient and stable up to 90 oC • With the low back pressure afforded by 5-µm 160 Å Fused-core

particles, columns of these particles are less prone to overpressurizing due to plugging and longer columns can be run for high resolution separations of proteomic samples

• With the correct choice of mobile phase, high resolution LC-MS data can be obtained for mAb separations using 400 Å Fused-core particles

Conclusions

Special thanks to Robert Moran for assistance with chromatographic measurements.

Acknowledgment