Improving characterization of monoclonal antibodies

77 Pages·2014·4.15 MB·English

Webinar Series
Improving characterization of
monoclonal antibodies
Making a better biotherapeutic
November 19, 2014
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Webinar Series
Improving characterization of
monoclonal antibodies
Making a better biotherapeutic
November 19, 2014
Brought to 
Participating Experts
you by the 
Science/AAAS 
Custom 
Publishing 
Niomi Peckham, M.Sc.
Office
Alexion Pharmaceuticals
Cheshire, Connecticut
I-Jane Chen, Ph.D.
PerkinElmer Sponsored by:
Hopkinton, MA
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EExxoo‐‐ggllyyccoossiiddaassee SSeeqquueenncciinngg UUssiinngg aa LLaabb‐‐
oonn‐‐aa‐‐CChhiipp FFoorrmmaatt
NNiioommii  PPeecckkhhaamm,,  SScciieennttiisstt,,  AAnnaallyyttiiccaall  SScciieenncceess
AAlleexxiioonn  PPhhaarrmmaacceeuuttiiccaallss,,  CChheesshhiirree  CCTT
Glycosylation of Antibodies
N-Linked

Oligosaccharide linked to
asparagine at Asn-X-Ser, Asn-
X-Thr, or Asn-X-Cys
O-Linked

Oligosaccharide linked to serine
or threonine
• (a) Introduction to Protein Structure: Second Edition, Branden & Tooze 
4
N‐Linked Oligosaccharides
Sources of heterogeneity
Bisecting GlcNAc

Asn
Galactosylation

Sialylation/Fucosylation

High Mannose
Sialic Acid 
Galactose
Commercial importance
N-acetylglucosamine
Bioactivity

Pharmacokinetics
Mannose 
Immunogenicity

Fucose
5
N‐Linked Oligosaccharide Structures
MAN‐5
G2F
Asn
Asn
G0F‐GN
G1F Asn
Asn (Biantennary 
Hybrid)
Galactose
N-acetylglucosamine
G0F
Mannose
Asn
Fucose
6
Caliper (PerkinElmer) LabChip® GXII
System Components –
• LabChip GXII Instrument
• LabChip GXII Software
• LabChip Reagent/Chips:
• DNA (1K, 5K, and 12K)
• RNA (100 – 6000 nucleotides)
• HT Protein (14 to 350+ kDa)
• HT Pico Protein (10 pg/μL – 100 ng/μL)
• N‐Linked Glycan Profiling
• Charge Variant (New! pI 7.0 to 9.5)
7
Caliper ProfilerPro™ Glycan Profiling Kit
• Analogous to HPLC or CE Glycan Profiling
• Complement to MALDI‐ToF‐MS for mass profiling
• Developed for neutral N‐linked glycans (e.g. Man5, G0F, G1F, G2F)
• Separation by molecular weight
• High Throughput – 96 well plate format, reagents pre‐aliquoted
• Measures relative amount of N‐Linked neutral glycans
• estimated LoQ of 1%
• Sample Requirements
• Minimum one step purified (e.g. Protein A), pH ~7.0
• Normalized to 5 mg/mL, as low as 2 mg/mL 
8
N‐Linked Oligosaccharide Profiling: Method
• Denaturation of protein with SDS‐βME solution at 70°C
• PNGase digestion removes N‐Linked glycans
• specifically cleaves the N‐acetyl glucosamine – asparagine linkage 
PNGase
Asn
• Label glycans with fluorophore
Dye
• Needed to visualize glycans
• Reductive amination
• Separate electrophoreticaly by size on chip
• Detect by laser induced fluorescence
9
Workflow of the Glycan Assay
Sample Preparation – < 4 hours
Chip Preparation ‐ < 15 minutes
Reconstitute  Clean chip  Place chip, ladder,  96 wells –
ladder and  and add gel  wash buffer and  separation in 
marker and marker samples in GXII ~1.5 hour
45 samples (duplicates) in < 6 hours 
10