Application note - Bruckner Analysentechnik · The purification of PEGylated proteins from the API and undesired multi-pegylated proteins is a challenging separation In the presented

© ChromaCon AG // Purification of PEGylated proteins with Contichrom® // www.chromacon.ch // ver. May 2013 1

Purification of PEGlyated proteins with Contichrom®

Application note


Summary

The purification of PEGylated proteins

from the API and undesired multi-

pegylated proteins is a challenging

separation

In the presented application example,

batch chromatography achieves for

the desired pegylated product at 93%

purity only yields of 56%

With Contichrom® equipment and the

MCSGP process, the yield can be

increased to 83%

Yield of PEGylated protein

50%

100%

Batch Contichrom®

56%

83%


Purification of PEGylated proteins

Model system: Separate PEGylated forms of

alpha-Lactalbumin with anion-exchange

chromatography

a) With batch chromatography

b) With Contichrom® in MCSGP chromatography mode


mPEG-SPA: Structure and Reactivity

Lysine residues attach to the activated PEG to

form the PEGylated protein

Hydrolysis half-life at pH 8, 25 °C, of mPEG-SPA is

16.5 min a

Nearly 80 % of the mPEG-SPA has reacted to give

PEGylated -Lactalbumin. The missing 20 %

mPEG-SPA may be breakdown product

hydrolysis

Roberts, M. J.; Bentley, M. D.; Harris, J. M. Advanced Drug Delivery Reviews 2002, 54, 459.


-Lactalbumin: Structure and Reactivity

Molecular weight 14.2 kDa

122 amino acids

12 lysine residues a

Isoelectric point 4.2-4.5 b

Hydrodynamic radius 2.0 nm c

a) Viaene, A.; Volckaert, G.; Joniau, M.; De Baetselier, A.; Van Cauwelaert, F. European Journal of Biochemistry 1991, 202, 471.

b) Browne, W. J.; North, A. C. T.; Phillips, D. C.; Brew, K.; Vanaman, T. C.; Hill, R. L. Journal of Molecular Biology 1969, 42, 65.

c) Smith M.; Handbook of Biochemistry, 2nd edn, Cleveland: The Chemical Rubber, 1970. p. C3–C292

Figure: PDB ID 1F6S


SEC chromatogram of PEGylated protein

Analytical HP-SEC chromatogram (Superdex 200

10/300, A280) from batch PEGylation of -

lactalbumin with mPEG-SPA 5 kDa in PBS (25 mM

Phosphate, pH 7.4)


SEC chromatogram of PEGylated protein

Analytical HP-SEC chromatogram from batch PEGylation

Product definition: The mono-PEGylated form P1 is the desired

species

P3 P2 P1 P0 M A

A: Aggregates

P3: tri-PEGylated

P2: di-PEGyated

P1: mono-PEGylated

P0: native protein

M: mPEG-SPA break-

down product


SDS-PAGE of SEC Fractions

a) Zheng, C. Y.; Ma, G.; Su, Z. ELECTROPHORESIS 2007, 28, 2801.

Non-reducing conditions a

Silver staining of purest PEGamer

fractions isolated from HP-SEC

Protein standards MW do not

correlate with PEGylated protein

Hydrodynamic radii

1 2 3 4 5 6 7

160 kDa

110 kDa

80 kDa

60 kDa

50 kDa

40 kDa

30 kDa

20 kDa

15 kDa

10 kDa

3.5 kDa

Lane 1: F25 – P0: native -Lactalbumin

Lane 2: F20 – P1: mono-PEGylated

Lane 3: F15 – P2: di-PEGylated

Lane 4: F13 – P3: tri-PEGylated

Lane 5: -Lactalbumin (MW 14.2 kDa)

Lane 6: concentrated Feed

Lane 7: Protein Standards

No

n-P

EG

Mo

no

-PE

G

Di-

PE

G

Tri

-PE

G

Fee

d

-L

ac

talb


Preparative AIEX Separation – batch

Linear gradient elution, single column, load 4.3 g/L

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

10 15 20 25 30 35 40 45 50 55

pu

rity

co

nc

[g

/L],

co

nd

[m

S/c

m]/

20

, p

H/1

0, A

28

0 [

mA

U]

sc

ale

d

time [min]

P1 P2

A280 cond

purity

Run #0613ARun

Pool fraction


Preparative AIEX Separation – MCSGP

Use Contichrom® software wizard to automatically convert batch chromatography

with linear gradient elution into MCSGP operating parameters

screenshot

Contichrom wizard

Recycle

P2/P1 target

pool P1

recycle

P1/P0

P0 Equilibration,

loading & washing


Preparative AIEX Separation – MCSGP

Operating point generated by wizard provides high

purity & yield U

V-s

ign

al (A

28

0)

reco

rde

d a

t P

-ou

tle

t Start-up Steady-state with stable performance


Preparative AIEX Separation – comparison

For the same purity, significantly higher yield is achieved with MCSGP

Desired

Product P1 Blue: Feed

(PEGylated protein mixture)

Red: MCSGP product pool

(corresponds to 83% P1 yield)

Green: Single column product pool

(corresponds to 56% P1 yield)


Preparative AIEX Separation – comparison

Batch

chromatography

Batch chromatography

MCSGP +10% purity

- 50% impurities

+30% yield


Summary

Separation of mono-pegylated protein from multi-

and un-pegylated protein is very challenging

Batch chromatography achieves only 56% yield

Batch can be automatically converted into MCSGP

operation using Contichrom® wizard

Performance improvements of MCSGP with

respect to batch:

+ 30% yield

-50% buffer consumption

Application note - Bruckner Analysentechnik · The purification of PEGylated proteins from the API and undesired multi-pegylated proteins is a challenging separation In the presented

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