© 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