QABio's Glycan Characterization Workshop Cambridge, Massachusetts

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QA-Bio's Glycan Characterization
WorkshopCambridge, Massachusetts

• Joseph Siemiatkoski
• Werner Meier
• 21 June 2004

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Assessing Structure-Activity Relationships (SARs)
of Glycoprotein Pharmaceuticals
The American Heritage Dictionary Relationship n.
1. The condition or fact of being related
connection or association Biogen
Idec Structure-activity relationship also SAR n.
1. The connection or association of a
physicochemical parameter with an in vitro
measure of activity or in vivo pharmacokinetic
behavior of an active pharmaceutical ingredient.
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A Partial Listing of Glycobiopharmaceuticals

• Activase (alteplase)
• Amevive (alefacept)
• Avonex (interferon ß-1a)
• Enbrel (etanercept)
• Fabrazyme (agalsidase ß)
• Refacto (antihemophilic factor)
• Herceptin (trastuzumab)
• Procrit (erythropoetin)
• Rituxan (rituximab)
• Zevalin (ibritumomab tiuxetan)
• And more on the way

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Why Determine Glycosylation Specific SARs?

• Determine pharmacological relevance of glycoform
  changes induced by
• Batch to batch variability
• Process changes (intended and unexpected- mfg
  site changes, raw material variability)
• Drug substance/drug product storage

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SAR Tools

• Generate glycovariants
• Characterize glycovariants biochemically
• MALDI
• ESI-MS of intact glycoproteins
• ESI-MS of permethylated glycans
• Chromatographic separation of glycans
• Characterize glycovariants biologically

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Generating Glycovariant Samples

• Glycovariant Sample Sources
• Manufacturing variability during development
• Glycosidase or glycotransferase modifications
• Chromatographic fractionation

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Biochemical Characterization of Glycovariants
Mass Spectral Techniques

• MALDI-TOF MS of released glycans

2053.22
1599.28
1760.12
1355.68
2037.40
1736.66
1379.72
1899.47
2062.33
2012.66
1926.66
1475.85

• ESI-MS of intact glycoprotein (IgG)

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Biochemical Characterization of Glycovariants

• Fluorescent labeling (2-AA) Anion-exchange
  chromatography

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Method Comparability, IgG Model
Total Galactose Residues Total sites for
Galactosylation

• Percent Galactosylation

G2
G1
G0
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Comparability of Method Precision
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Method Linearity Experiment
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Agreement of Analytical Methodologies
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Biological Characterization

• Binding assays
• In vitro activity assays
• Cytopathic effect
• Cell to cell bridging
• Effector function
• Pharmacokinetic studies
• In vivo efficacy studies

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4 Case Studies
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Model Glycoprotein 1Predominantly
Tetra-antennary glycans

• Receptor-IgG1
• Six N-linked sites in ectodomain
• (Bi 20, Tri 19, Tetra 41)
• Conserved N-linked sites in Fc

Question What impact does changing sialylation
have on the properties of a predominantly
tetra-antennary glycoprotein? Approach
Prepare 2,6-sialyltransferase treated material
and assess bioactivity and pharmacokinetic (PK)
properties.
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Altering sialylation has significant effects on
the PK properties of glycoprotein 1

• The PK properties of two preparations with
  relative sialylation levels of 78 and 95 were
  tested in mice.
• An approximately 22 increase in sialic acid
  content increases the T ½ by 3.4-fold.
• In vitro biological activity appears unaffected
  by increased sialylation

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Model Glycoprotein 2Predominantly Biantennary
Glycans

• Receptor-IgG1
• Four N-linked sites in ectodomain
• (Bi 58, Tri 5, Tetra 2)
• Conserved N-linked sites in Fc

Question What impact does changing sialylation
have on the PK properties of a predominantly
biantennary glycoprotein? Approach Prepare
neuramindase and 2,6-sialyltransferase treated
material and assess PK properties.
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Altering Sialylation has little effect on the PK
Properties of Glycoprotein 2

• The PK properties of three preparations with
  relative sialylation levels of 11, 46, and 74
  were tested in mice.
• An approximately 85 reduction in SA content does
  not significantly affect the T ½.

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Model Glycoprotein 3O-linked glycans

• Receptor-IgG1
• Up to twelve O-linked sites in ectodomain
• (mono and disialylated core-1 structures)
• Conserved N-linked site in Fc

Question What impact does changing sialylation
on O-linked glycans have on PK and
activity? Approach Prepare neuramindase
treated material and assess bioactivity and PK
properties.
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Reducing O-sialylation has no effect on in vitro
activity but significantly alters PK properties
of glycoprotein 3
Sialic acid level was reduced by approximately 90
-
Relative inhibitory activity of desialylated
model protein 3
Pharmacokinetic parameters of desialylated
glycoprotein 3
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PK Parameter
Control
Desialylated
65
35.6
139
t1/2, h
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T ½ is reduced 4-fold.
45
35
25
15
0.01
0.1
1
10
Concentration (Log nM)
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Model Glycoprotein 4

• IgG1
• conserved N-linked site in Fc
• approximately 35 galactosylation

Question What impact does variability of Fc
glycosylation have on bioactivity? Approach
Prepare range of glycodistributions by
exoglycosidase treatments (galactosidase and
glucosaminidase), determine CD16 binding in
bridging format bioassay.
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Extensive Degradation of Glycans Required to
Produce Significant Effect in CD16 Binding
CD16 (Jurkat)/Target Cell Bridging Activity

• Assay Control
• Mock Treated
• Galactosidase Treated
• Galactosidase/ Glucosaminidase Treated

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Summary

• The sialylation level of N and O-linked glycans
  can significantly contribute to changes in PK
  properties.
• Small differences in sialylation of
  tetra-antennary N-linked structures can lead to
  large reduction of T ½
• Large differences in sialylation of biantennary
  N-linked structures have less effect on T ½
• Reducing sialylation on O-linked structures led
  to large reduction in T ½
• Antennary distribution for the model
  glycoproteins studied did not have direct effects
  on in vitro bioactivity.
• Any effects of glycosylation variability on
  product safety would be assessed as part of a
  preclinical or clinical trial separate from the
  SAR studies.

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Conclusions

• Glycosylation is a potential hot spot in a
  molecule, which can influence pharmaceutical
  behavior. It is prudent to allocate resources to
  understand structure-activity relationships early
  in development.
• Extent of effect of variations in glycosylation
  is glycoprotein specific, it is important to
  empirically establish the potential biological
  effects.
• Examine effects of glycosylation parameters
  (e.g. sialylation or antennary distribution)
• Assemble SAR data package to guide product
  development and specifications

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References

• Baenziger, J. U., Maynard, Y. (1980) Human
  Hepatic Lectin Physicochemical Properties and
  Specificity. J. Biol. Chem., 255, 4607-4613.

Animal studies were approved by an Institutional
Animal Care and use Committee.
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Glycoanalysis Benchmarking Exercise

• Two test articles
• An immunoglobulin
• A sialylated serum protein
• Determine glycodistribution in each participating
  lab, using their assays
• Compare results obtained
• For precision and accuracy
• Across assay types
• Organized by Richard Cummings, Ph.D. Director,
  Oklahoma Center for Medical Glycobiology and J.
  Siemiatkoski of Biogen Idec