© 2006

WCBP 2006

WCBP 2006

Assessing Protein Aggregation

by FFF-MALS Technique

Michelle Chen, Dierk Roessner, John Champagne

Wyatt Technology Corporation

6300 Hollister Ave.

Santa Barbara, CA 93117

© 2006

WCBP 2006

Eclipse FFF – MALS System

FFF: field flow fractionation; MALS: multi-angle light scattering

© 2006

WCBP 2006

Limitation of SEC Separation for Protein Aggregation

• Absent of interaction between protein & column packing

• Wider separation and protein concentration range

• More mobile phase choices, including formulation buffer.

• Column packing may remove part of aggregates.

• Column shearing may alter aggregation.

• Limited choices of mobile phase.

Advantages of using FFF as an Alternative?

© 2006

WCBP 2006

A Stressed Antibody Analyzed by FFF-MALS

MW determined by MALS

LS traceUV trace

© 2006

WCBP 2006

Same Stressed Antibody Analyzed by SEC-MALS

Less resolution for high order of oligomers

© 2006

WCBP 2006

Antibody Sample with Dimer and Fragments

MW measured by MALS

© 2006

WCBP 2006

Antibody Sample with Large Aggregates

Large aggregates may be removed by a column

© 2006

WCBP 2006

Antibody Exhibiting Opalescence

A B C D

A: Formulation Buffer (with foam)

B: IgG at ~60 mg/mL (Release Clarity = I % Monomer >99%)

C: EP Standard II

D: EP Standard III

© 2006

WCBP 2006

2 4 6 8 10 12 14

0.00

0.75

1.50

2.25

3.00

3.75

c(s 2

0,w)

[s20,w

] Svedbergs

0.113 mg/mL 0.420 mg/mL 0.936 mg/mL

AUC Results Do Not Explain Opalescence

Samples appeared very homogeneous with only ~1 % HMW.

© 2006

WCBP 2006

FFF -MALS Reveal the Opalescence Source …

Unknown species appears to have a mass of over 100 MDa!

© 2006

WCBP 2006

FFF-MALS Can Be Fully Validatedmolar mass vs. time/volume

BSA 1mgmL 60uL 490um 3zu1 05.vafgfedc BSA 1mgmL 60uL 490um 3zu1 07.vafgfedcb BSA 1mgmL 60uL 490um 3zu1 08.vafgfedcbBSA 1mgmL 60uL 490um 3zu1 09.vafgfedcb BSA 1mgmL 60uL 490um 3zu1 10.vafgfedcb BSA 1mgmL 60uL 490um 3zu1 11.vafgfedcbBSA 1mgmL 60uL 490um 3zu1 12.vafgfedcb BSA 1mgmL 60uL 490um 3zu1 13.vafgfedcb BSA 1mgmL 60uL 490um 3zu1 14.vafgfedcb

time or volume15.0 20.0 25.0 30.0 35.0 40.0

mo

lar

mas

s (g

/mo

l)

51.0x10

61.0x10

71.0x10

81.0x10

91.0x10

101.0x10

© 2006

WCBP 2006

FFF-MALS Reproducibilityrun monomer monomer dimer dimer higher higher total

µg % µg % µg % µg

1 38,82 70,21 9,60 17,36 6,69 12,11 55,29

2 39,05 69,90 9,70 17,36 6,97 12,47 55,86

3 39,23 70,23 9,66 17,30 6,78 12,14 55,86

4 39,07 69,91 9,68 17,32 6,89 12,32 55,89

5 35,47 70,02 8,78 17,34 6,13 12,11 50,66

6 38,37 68,73 9,88 17,70 7,28 13,04 55,84

7 38,92 69,01 9,99 17,71 7,28 12,90 56,39

8 39,46 69,52 9,93 17,50 7,09 12,50 56,76

9 39,65 69,91 10,00 17,63 6,85 12,07 56,71

10 39,85 69,99 9,90 17,38 6,84 12,02 56,93

11 40,02 69,88 10,02 17,50 7,00 12,22 57,27

12 40,14 69,88 10,06 17,51 7,02 12,22 57,45

13 40,23 69,94 10,07 17,50 7,05 12,26 57,52

14 40,01 70,08 9,88 17,30 6,90 12,09 57,09

average 39,16 69,80 9,80 17,46 6,91 12,32 56,11

std deviation 1,16 0,42 0,32 0,14 0,27 0,30 1,65

RSD% 3.0 3.3 3.9 3.0

Change of membrane