DLS distributions in once freeze dried samples of IgG

- DLS data for once freeze IgG without added excipient.- Rh for IgG ~ 5.51nm. [Thermal Properties of human IgG, (1987) Molecular Immunlogy, 24:495-501]- The DLS data is consistent with this

[A] [B]

[C]

Intensity Distribution

Number Distribution

Mass Distribution

Rh = 5.2nm

Rh = 5.2nm

IgG

0

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30

Number Distribution0 IgG1FDC2FDC3FDC4FDC5FDC6FDC7FDC

S

ize (n

m)

IgG0

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30Mass Distribution

0 IgG1FDC2FDC3FDC4FDC5FDC6FDC7FDC

S

ize (n

m)

- Influence of successive freeze drying cycles on the IgG sample without added excipients - The size of IgG aggregates increases with repeated freeze drying cycles.

[A] [B]

IgG + 1.8mM Sucrose

IgG + 3.6mM Sucrose

0

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25Number Distribution

0FDC

1FDC

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3FDC

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ize (n

m)

IgG + 1.8mM Mannitol

IgG + 3.6mM Mannitol

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Number Distribution0FDC

1FDC

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3FDC

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ize (n

m)

Effect of the concentration of sucrose and mannitol on the aggregation of IgG

- Influence of sucrose during successive freeze drying cycles on the size of IgG

- the aggregation of IgG is less pronounced for the higher concentration of sucrose

- Influence of mannitol during successive freeze drying cycles on the size of IgG

- Mannitol appears to promote aggregation of IgG, and is more pronounced at the higher concentration

AFM Study of the Effect of Multiple freeze drying on IgG

- AFM images reveal both crystalline and amorphous features. Globular, protein like features can also be observed. - The size of the smaller globular features is similar to the size of monomeric IgG (~15-20nm) reported in previous AFM studies (Ultramicroscopy 105:103-110). The larger globular features likely represent aggregates of IgG

250nm 250nm 250nm

Cycle 1 Cycle 2 Cycle 3

AFM images of freeze dried IgG samples, prepared without excipients (starting concentration 2mg/ml in 0.01M PBS)

Globular features

Crystalline

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AFM study of IgG Freeze Dried with 20mM Sucrose

- AFM images predominantly show amorphous regions with associated globular features, again most likely due to IgG monomers and aggregates. Crystalline features were rarely observed.

250nm 250nm 250nm

250nm

A B C

D

Amorhous

Crystalline

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AFM study of IgG Freeze Dried with 20mM Mannitol

- AFM images show both amorphous and crystalline features. - Globular features appear to be associated with distinct regions on the sample surface e.g. some crystalline features do not appear to be coated with a protein like layer.

250nm250nm 250nm

A B C

150 nm

D

Crystalline Amorhous

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IgG Freeze Dried with Sucrose and Mannitol in Combination [2]

-Images of once freeze dried IgG with Sucrose (40mM) and Mannitol (20mM) in combination. - An increase in the molar concentration of sucrose predominantly reveals amorphous features - Again globular features are associated with these regions.

250 nm 250 nm 250 nm

150 nm

A B C Amorphous