IDEXX Laboratories, Inc. - PerkinElmer

IDEXX Laboratories, Inc. 

Antibody Assault: How CE Helped 

Uncover Processing Challenges 

Kristin Riley, Research Scientist 

Perkin Elmer Users Group Meeting 

6/7/12

2 | © 2012 IDEXX Laboratories, Inc. All rights reserved.

Reliable Science for Better Medicine 

• More than 350 scientists, engineers and veterinarians on staff 

worldwide 

• 50+ PhDs in biochemistry, cell biology, immunology, molecular 

biology, physical chemistry, physics and microbiology 

• 130+ specialists and 90+ board-certified veterinarians 

• Diverse technologies 

• Immunoassays 

• DNA probes 

• Clinical chemistries 

• Hematology 

• Molecular and cellular biology 



2011 Revenue: $1.219 billion 


Companion Animal Group 

Diagnostics and information technology solutions; 2011 Revenue: $1.0 billion 


Rapid Assays: Point-of-care SNAP ® kits 

Market leader: Canine • Feline • Equine 

• Family of proprietary SNAP ® tests 

• SNAP ® 4Dx ® Plus Test, 

launched in April, detects 

six vector- and tick-borne 

diseases in one SNAP kit 

• Client value with pet-owner 

reporting 



Rare Reagent tPilot tPlant tCOE 

• Incorporate Cycles of Learning on Process Development and Product 

Support 

• Mitigate risk earlier in the product development lifecycle 

• Apply rational design and structure/functional relationships to 

reagents 


What do we do? 

• Scale up and transfer robust processes for reagent 

manufacture 

• Analytical Characterization 

mAU 

15.49 

30.0 

12.96 

25.0 

HRPO Incorporation Ratio = 0.72 

20.0 

15.0 

10.0 

11.18 

11.11 15.51 

5.0 

15.49 

0.0 

9 | © 2012 IDEXX Laboratories, Inc. All rights reserved. 

0.0 5.0 10.0 15.0 20.0 25.0 ml

The Tool Box 

•Process Scale up: 

• Sciclone G3 (use with PhyNexus tips) 

• Chromatography 

• Particle Coating 

• Conjugations 

• Purifications 


The Tool Box 

• Analytical Characterization: 

• SEC/MALS 

• CE 

• LC/MS 

• RP-HPLC 

• DLS 

• FT-IR 

• TGA 

• Outsourced: NMR, OR, Elemental analysis, 2D gels etc. 


Antibody-Enzyme Bioconjugates for 

Immunoassays 

ENZ 

NH 2 

ENZ 

H 

N 

O 

O 

N 

O 

HS 

SH 

O 

O 

O 

N 

O 

O 

N 

O 

ENZ 

H 

N 

O 

O 

N 

O 

S 

Sulfo-SMCC 


• IgYs are ~180 kD. 

IgY Antibodies 

• There are many (>>10) disulfide bonds inter and intra chain(s) in an IgY. 

• The MW differential (relative to an IgG) is in the heavy chain (Fc) 

IgG 

IgY 


Wilmar Dias da Silva, Denise V. Tambourgi. Veterinary Immunology and 

Immunopathology 135 (2010) 173–180.

Process Map for Conjugation 

Activate Enzyme 

Thaw and 

Reduce Antibody 

Purify Ab from 

Reductant 

Incubate with 

Enzyme 

Analytical 

Analysis 

Storage for use 

in Product 


Characterization by Analytical SEC 

mAU 

Conj 40.0 

( 30.00 

20.0 

(A280) 

Conj (A403) 

Ab (A280) 

ENZ (A280) 

ENZ (A403) 

• Advantage: 

• Use “ENZ incorporation 

ratio” (Area 403 / Area 280) 

to provide spec for 

predictive success of 

conjugate. 

10.0 

• Disadvantage: 

• Method resolution is 

not optimal for individual 

species. (Limited by 

column choice) 

0.0 

0.0 5.0 10.0 15.0 20.0 25.0 ml 


Characterization by Analytical SEC 

mAU 

200 

Non-Reduced Ab 

150 

shown in orange 

Reduced Ab 

shown in green 

100 

50 

0 

0.0 5.0 10.0 15.0 20.0 25.0 ml 


Non-Reduced IgY Ab (in non-reducing CE conditions) 

Intact Ab 

System 

Peak 

Reduced IgY Ab (in non-reducing CE conditions) 

Light 

Chain 

Heavy 

Chain 

? 


Characterization by Analytical CE 

Overlay: Non-Reduced and Reduced Ab 

Blue=Non- 

Reduced Ab 

Red=Reduced 

Ab 


Enzyme in non-reducing conditions 

ENZ 

Conjugate in non-reducing conditions 


Conjugate and Components by CE 

Conclusions 

• We have limited or no selectivity in disulfide reduction. Most of the IgY S-S is broken by 

the reduction. 

• We are relying on hydrophobic, H-bond and ionic interactions to maintain IgY structural 

integrity. 

• Of the 7 ENZ containing IgY conjugate species in CE only four contain active light chain. 

• Free, unconjugated light chain is a significant fraction 

• Conjugated heavy-chain-only species are a significant fraction 


The Rescue 

• Metal ions have been shown to catalyze disulfide formation by oxidation of thiols 


Process Map for Conjugation 

• Metal ions were added to final conjugate to oxidize disulfides and stabilize Ab 

Activate Enzyme 

Thaw and 

Reduce Antibody 

Metal Ions added, 

ON incubation 

Purify Ab from 

Reductant 

Incubate with 

Enzyme 

Analytical 

Analysis 


Storage for use 

in Product

Non-Reduced IgY Ab (in non-reducing CE conditions) 

Reduced IgY Ab (in non-reducing CE conditions) 

Light 

Chain 


Control Conjugate 

Free ENZ 

Conjugate 

Species 

Free Light Chain 

Conjugate in presence of 5X Cu(II) ions 


Control Conjugate 

Conjugate in presence of non-catalyzing M+ ion 



Global l leader 

• A portfolio of businesses with 

global market leadership 

• Pet healthcare technology 

• Production animal diagnostics 

• Water microbiology 

• Strategy of innovation in diagnostic 

and information technology 

• Track record of growth and 

profitability 


Next tSteps 

• Functional Analysis 

• Study other conjugates 

• Glycan analysis 


Thank you! 

Extended Team Members: 

Kaitlin Lawlor 

Chris Stetson 

Rudy Groat 

Julie Cross

IDEXX Laboratories, Inc. - PerkinElmer

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