Aggregate Analyses by Light Scattering Complementary and OrthogonalSigrid Kuebler, Ph.D, Dan Some, Ph.D, and Jeff Ahlgren, Ph.D. — Wyatt Technology

Introduction
Technique

Abstract: Biotherapeutic regulatory filings
require detailed characterization ofdegradation pathways including aggregation.
Three orthogonal light scattering techniques
complement each other to analyze andquantify soluble and sub-micron insoluble
aggregates: SEC-MALS, FFF-MALS and CGMALS. This poster describes the capabilitiesof each technique and how they work together
to provide a comprehensive picture ofirreversible and reversible aggregates.

SEC-MALS
0.5 – 20 nm(column dependent)

Separation size
range

FFF-MALS

CG-MALS

1 nm – 1 µm

noneProperties
determined

Quantitative distributions ofabsolute MW and size (Rg, Rh)

Quantitative distributions of absolute
MW and size (Rg, Rh)

Average MW and size;A2(B22), self / hetero-association Kd,
co-solute interaction A11

Resolution

Proteins, fragments, small
aggregates (column dependent)

Proteins, fragments, soluble andnon-soluble aggregates

Stoichiometry and association propertiesof reversible aggregates and complexes

Column (high surface area)

Open channel or hollow fiber(low surface area)

noneSample dilution

Concentrates samples during
focusing step; dilutes after elution

none

Separation matrix
Change inconcentration

SEC-MALS: absolute molar mass of proteins,oligomers and fragments

FFF-MALS: quantify soluble and insoluble aggregates
Cross Flow

• Any leading HPLC-SEC for
separation and UV detection

Diffusion

Parabolic
Channel Flow

• High-performance WTC-030S5
SEC column• Eclipse FFF + HPLC for
separation and UV detection• DAWN or miniDAWN MALSdetector• DAWN or miniDAWN MALSdetector• Optilab RI detector• Optilab RI detector

Trace Aggregates

1x106

1x105

1x108

MW determined byMALS

LS trace

1x105

UV trace

Molar Mass (g/mol)

Molar Mass (g/mol)

Molar Mass (g/mol)

Relative Scale

1x106

LS…and all sizes of aggregates

Resolve and analyze oligomers…

37° C for 6 months

Conc.

FritMembrane

1x106

Large aggregates that may beremoved by SEC columns
1x104

1x104
101214161810201214161820

10Time (min)Time (min)LS is more sensitive to high-MW
fractions than concentration detectors

MALS + RI determines absolute molar
masses of known and unknown proteins•

141618• Optilab RI detector

1416

Eclipse FFF is a recognized orthogonaltechnique to SEC for quantifying oligomers

Conclusions
Orthogonal and complementary

SEC-MALS with a DAWN
provides:•• Quantitative distributions of absolute•

Mutually orthogonal biophysicaltechniques for characterizing molar
mass, size and aggregates

molar mass and size

• Identification of fragments and foreign• Characterization of conjugates such

• Validate overall SEC-MALS or
FFF-MALS Mw to determine ifaggregates have been filtered outor disrupted.

FFF-MALS with an Eclipse andDAWN provides:

CG-MALS with a Calypso and
DAWN provides:• Orthogonal approach to SEC-MALS• Indication of aggregate loss in SEC or• Identify reversible aggregates at
actual formulation concentrations• A wider range of separation than SEC• Compare lots in terms of weightaverage molar mass and proteinprotein interactions (A2 / B22).

• Characterization of analytes too large

proteins as well as oligomers
as PEGylated or glycosylated proteins

Automated CG-MALS measurements:

2.5x105

90° LS and dRI (arb. Units)

2x105

1.5x105

1x105

0.5x105

0101214161816Time (min)Lot 1 Mw

Lot 2 Mw

SEC-MALS

151.5  1.8 kDa

150.8  2.2 kDa

CG-MALS

150.2  2.0 kDa

149.0  1.2 kDa

18• Highlight differences in A2 thatindicate changes in surface
residues (e.g. deamidation) or
higher-order structural shifts.

18

Large aggregates that may be removed or
disrupted by SEC columns remain intact
and are characterized in FFF

Comparison of results from thesecomplementary techniques identifies
aggregate properties and behavior.

Lot-to-lot comparability and SEC-MALS validation

Molar Mass (g/mol)

12Time (min)system• DAWN or miniDAWN MALS
detector

1020Time (min)CG-MALS: validate overall MW and protein-protein
interactions
Calypso composition-gradient

12for characterizing biopharmaceuticals.
with no shear, less dilution and little
surface interactionsfor SEC: protein-polysaccharide
conjugate vaccines, lipoproteins,liposomes, viruses and virus-likeparticles.

FFF• Measures of protein-protein
interactions including self-associationand co-solute interactions• A quick, simple way to compare lots