Platzhalter für Bild, Bild auf Titelfolie hinter das Logo einsetzen

Julia Kahle, Holger Zagst, Kai Jorrit Maul-Köhler, Rebecca Wiesner, Matthias Stein and Hermann Wätzig

CE Pharm, September 12th 2018 in San Francisco

Size- and charge-based Separations for Biopharmaceutical Analysis

Comparative Performance Studies with various CE and HPLC techniques

Julia Kahle | CE Pharm 2018 | 2

Characterization of biopharmaceuticals

Size variants Charge variants

CE CE-SDS

MCE-SDS

CZE

cIEF

icIEF

HPLC SEC

iSEC

AEX

CEX

CE-SDS

MCE-SDS

SEC

MEKC

icIEF

cIEF

CEX

Julia Kahle | CE Pharm 2018 | 3

Size heterogeneity – Experimental design

Test conditions

• Established reference methods with

only few method adjustments

• Protein mixture A and B

• Preparation of stock solutions,

stored at -30°C

Conventional parameters Additional parameters

Resolution Ease of use

Precision Flexibility

Linearity Reliability

Sensitivity Sustainability

Determination of performance parameters:

Test molecules

• Aprotinin 6.5 kDa

• Lysozyme 14.4 kDa

• Myoglobin 17.8 kDa

• Carbonic anhydrase 29 kDa

• Ovalbumin 42 kDa

• BSA 66 kDa

Kahle, J., Maul, K. J., Wätzig, H., Electrophoresis 2018, 39, 311–325. https://doi.org/10.1002/elps.201700278.

Julia Kahle | CE Pharm 2018 | 4

0

20

40

60

80

100

14 16 18 20 22 24

Absorp

tion

[m

AU

]

Migration time [min]

RSD values (ovalbumin, n=40)

tR = 0.096%; peak area = 2.1%

SEC and CE-SDS – Peak profiles of protein mixtures

Size exclusion chromatography

Capillary electrophoresis-SDS RSD values (ovalbumin, n=60)

tM = 0.35%; peak area = 10%

• VWR Hitachi LaChrom Elite

• Yarra 3u SEC-2000, 300 x 7.80 mm

• 50 mM Na2HPO4, 300 mM NaCl, pH 6.8

• 1.0 ml/min, UV abs. at 220 nm

• Agilent 7100 CE System

• fused silica, Le 24 cm, Ø 50 µm

• 100 mM Tris, 1% SDS (m/m), pH 8.0

• -16.5 kV, UV abs. at 220 nm

• method based on Cianciulli et al. (2012)*

*see references

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-5

0

5

10

15

20

25

0 0.5 1 1.5

Ab

so

rpti

on

[m

Au

]

Relative Migration Time

Maurice S. (Mixture B)

Myoglobin Carbonic anhydrase Ovalbumin BSA

-100

150

400

650

900

1150

1400

0 5 10 15 20 25

Flu

ore

sc

en

ce

Aligned Time (sec)

LabChip® GXII Touch HT (Mixture B)

Myoglobin Carbonic anhydrase Ovalbumin BSA

0

30

60

90

120

150

180

0 5 10 15 20 25 30

Ab

so

rpti

on

[m

Au

]

Time [min]

PrinCE NextI870 (Mixture A)

Aprotinin Lysozyme Carbonic anhydrase

0

55

110

165

220

275

330

0 5 10 15 20

Ab

so

rpti

on

[m

Au

]

Time [min]

Agilent 7100 CE System (Mixture A)

Aprotinin Lysozyme Carbonic anhydrase

CE-SDS – Peak profiles of protein mixtures

• Le 24.5 cm, Ø 50 µm, fused silica

• UV abs. at 200 nm

• 100 mM TRIS, 1.0% SDS (m/m),

10 mM DTT, pH 8.0

• HT Protein QC Assay

• Le 14 mm, fused silica

• LIF-Ex/Em 635/700 nm

• CE-SDS Size Application Kit

• Le 15 cm, Ø 50 µm, fused silica

• UV abs. at 220 nm

• Le 26 cm, Ø 50 µm, fused silica

• UV abs. at 200 nm

• 100 mM TRIS, 1.0% SDS (m/m),

10 mM DTT, pH 8.0

Kahle, J., Maul, K. J., Wätzig, H., Electrophoresis 2018, 39, 311–325. https://doi.org/10.1002/elps.201700278.

Julia Kahle | CE Pharm 2018 | 6

0

10

20

30

40

50

0 40 80 120 160 200 240 280 320

Are

a [%

]

Run

LabChip® GXII Touch HT (Mixture B)

Carbonic anhydrase Myoglobin BSA Ovalbumin

0

10

20

30

40

50

0 30 60 90 120

Are

a [%

]

Run

Agilent 7100 CE System (Mixture A)

Aprotinin Carbonic anhydrase Lysozyme

0

10

20

30

40

50

0 10 20 30 40 50

Are

a [%

]

Run

Maurice S. (Mixture B)

Carbonic anhydrase Myoglobin BSA Ovalbumin

0

10

20

30

40

50

1 2 3 4 5 6

Are

a [%

]

Run

PrinCE NextI870 (Mixture A)

Carbonic anhydrase Aprotinin Lysozyme

CE-SDS – Intermediate precision of %peak areas

RSD (CA) = 1.7% RSD (CA) = 2.8%

RSD (CA) = 4.0%

RSD (CA) = 1.2%

4 preparations of each 30 1 preparation of 6

8 preparations of each 40 2 preparations of 30 and 22

Kahle, J., Maul, K. J., Wätzig, H., Electrophoresis 2018, 39, 311–325. https://doi.org/10.1002/elps.201700278.

Julia Kahle | CE Pharm 2018 | 7

Charge heterogeneity – Experimental design

Test conditions

• Established reference methods

• Preparation of stock solutions

Conventional parameters Additional parameters

Resolution Ease of use

Precision Flexibility

Linearity / Sensitivity Reliability

Determination of performance parameters:

Test molecules

• NISTmAb pI ≈ 9.2

• Infliximab pI ≈ 7.6

In preparation: Kahle, J., Zagst, H., Wiesner, R., Wätzig, H., Electrophoresis (2018).

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8.9 9.0 9.1 9.2 9.3

0

100

200

300

400

Absorp

tion [m

Au]

pI

8.0 8.2 8.4 8.6 8.8 9.0

0

20

40

60

80

Absorp

tion [m

Au]

tM [min]

17 18 19 20 21 22 23

0

100

200

300

Absorp

tion [m

Au]

tR [min]

35.5 36.0 36.5 37.0 37.5

0

20

40

60

80

100

120

Absorp

tion [m

Au]

tM [min]

Resolution – NISTmAb

I. icIEF (Maurice C.)

II. CEX (LaChrom Elite)

III. MEKC (7100 CE System)

IV. cIEF (7100 CE System)

*see references

Le 40 cm, Ø 50 µm, fused silica

UV abs. at 214 nm

400 mM EACA, 2 mM TETA,

Polysorbate-20 0.03%, pH 5.7

method: Turner and Schiel (2018)*

Le 24.5 cm, Ø 50 µm, LPA-coated

UV abs. at 280.2 nm / Ref 550.5 nm

PL 8-10.5 3%, PL 3-10 1%,

2.4 M urea, 40 mM L-Arg, 2 mM IDA

YMC-BioPro SP-F, 5 µm

UV abs. at 215 nm

20 mM phosphate buffer, pH 6.7

salt gradient with 0.5 M NaCl

method: Michels et al. (2015)*

Le 5 cm, Ø 100 µm, FC-coated

UV abs. at 280 nm

PL 8-10.5 4%,

3 M urea, 0.35% MC

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17 18 19 20 21 22 23

0

100

200

300

Absorp

tion [m

Au]

tR [min]

8.9 9.0 9.1 9.2 9.3

0

100

200

300

400

Absorp

tion [m

Au]

pI

Amounts of charge variants

icIEF cIEF MEKC CEX

Main 53% 55% 72% 74%

Basic 10% 11% 13% 17%

Acidic 37% 34% 14% 9.1%

NISTmAb

icIEF

CEX (LaChrom Elite) icIEF (Maurice C.)

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7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0

0

20

40

60

80

100

120

Absorp

tion [m

Au]

pI

11 12 13 14

0

5

10

15

20

25

30

35

Absorp

tion [m

Au]

tM [min]

11 12 13 14 15 16 17 18 19 20

0

25

50

75

100

125

150

Absorp

tion [m

Au]

tR [min]

39.5 40.0 40.5

0

20

40

60

80

100

120

140

Absorp

tion [m

Au]

tM [min]

Resolution – Infliximab

Le 5 cm, Ø 100 µm, FC-coated

UV abs. at 280 nm

PL 5-8 2%, PL 8-10.5 2%,

3 M urea, 0.35% MC

Le 24.5 cm, Ø 50 µm, LPA-coated

UV abs. at 280.2 nm / Ref 550.5 nm

PL 5-8 1.5 %, PL 8-10.5 1.5%,

2.4 M urea, 40 mM L-Arg, 2 mM IDA

YMC-BioPro SP-F, 5 µm

UV abs. at 214 nm

10 mM MES, pH 6.0

salt gradient with 0.1 M NaCl

method: Goyon et al. (2017)*

*see references

I. icIEF (Maurice C.)

II. CEX (LaChrom Elite)

III. MEKC (7100 CE System)

IV. cIEF (7100 CE System)

Le 40 cm, Ø 50 µm, fused silica

UV abs. at 214 nm

400 mM EACA, 2 mM TETA,

Polysorbate-20 0.03%, pH 5.7

method: Turner and Schiel (2018)*

Julia Kahle | CE Pharm 2018 | 11

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60

%A

rea

Injection number

CEX (VWR Hitachi LaChrom Elite)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60

%A

rea

Injection number

MEKC (Agilent 7100 CE System)

0

10

20

30

40

50

60

0 10 20 30 40 50 60

%A

rea

Injection number

icIEF (Maurice C.)

Control charts – NISTmAb %Areas

RSD (Main Peak) = 1.3%

RSD (Main Peak) = 1.5% RSD (Main Peak) = 1.5%

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D-Optimal design – NISTmAb

Parameter Minimum Mean Maximum

Pharmalyte conc. 2.0% 5.0% 8.0%

Pharmalyte ratio

(8-10.5+3-10) 1+1 3+1 1+0

L-Arginine 0 mM 5 mM 10 mM

Urea 2 M 3 M 4 M

1. Definition of the experimental space

2. Choice of responses

o e.g. resolution, pI value, pixel position, focusing time

3. Performance of measurements

o 20 different parameter combinations, each n=6

4. Data evaluation

o response Y = design matrix X * coefficient vector β + statistical error e

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Resolution:

2.30774 [2.23559; 2.38222]

2

2.05

2.1

2.15

2.2

2.25

2.3

2.35

2.4

2.45

2.5

1 2 3 4 5 6 7 8 9 3 4 5 6 7 55 60 65 70 75 80 85 90 95 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8

4.75 5 75 2.9

Predicted response graph – Resolution

L-Arginine [mM] PL total conc. [%] PL 8-10.5 [%] Urea [M]

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Resolution:

2.30774 [2.23559; 2.38222]

2

2.05

2.1

2.15

2.2

2.25

2.3

2.35

2.4

2.45

2.5

1 2 3 4 5 6 7 8 9 3 4 5 6 7 55 60 65 70 75 80 85 90 95 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8

4.75 5 75 2.9

Predicted response graph – Resolution

L-Arginine [mM] PL total conc. [%] PL 8-10.5 [%] Urea [M]

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Response surface plots – Resolution

Resolution value:

basic peaks

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Take Home Messages

o CE-based techniques show a much higher separation efficiency,

but in some cases a lower precision compared to HPLC.

o Depending on the intended application, each user can find the

most appropriate instrument for CE-SDS.

o An excellent performance was confirmed for icIEF, cIEF and MEKC.

o Design of Experiments is a powerful tool.

Julia Kahle | CE Pharm 2018 | 17

Thank you very much!

Research group of Prof. Dr. Hermann Wätzig

Kai Jorrit Maul-Köhler was involved in the CE-SDS experiments, Holger Zagst and Rebecca Wiesner

in the charge-based experiments and Matthias Stein in the DOE approach.

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Thank you very much!

• Thanks to Protein Simple and Perkin Elmer for providing their instruments and in

particular Susanne Dörks, Udo Burger and Xin Jiang from Protein Simple for the

tremendous support.

• Special thanks to the CASSS committee for the Student Travel grant and the

opportunity for scientific exchange.

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References

• Cianciulli, C., Hahne, T., Wätzig, H., Electrophoresis 2012, 33, 3276–3280.

• Kahle, J., Maul, K. J., Wätzig, H., Electrophoresis 2018, 39, 311–325.

• Turner, A., Schiel, J. E., Anal Bioanal Chem, 2018, 410, 2079–2093.

• Michels, D. A., Ip, A. Y., Dillon, T. M., Brorson, K., Lute, S., Chavez, B., Prentice, K. M.,

Brady, L. J., Miller, K. J., in: Schiel, J. E., Davis, D. L., Borisov, O. V. (Eds.), ACS

Symposium Series, American Chemical Society, Washington, DC, 2015, 237–284.

• Goyon, A., D'Atri, V., Bobaly, B., Wagner-Rousset, E., Beck, A., Fekete, S., Guillarme,

D., J Chromatogr B, 2017, 1058, 73–84.