In Vitro Glycoengineering - Its Application and Effect on IgG1

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1 In Vitro Glycoengineering - Its Application and Effect on IgG1 Glycobiology Conference, 2015 AAPS National Biotechnology Conference, June 8-10, 2015, San Francisco Dietmar Reusch, Marco Thomann, Development Analytics, Roche Diagnostics GmbH, Germany

2 How to Investigate the Impact of an Individual Fc Glycan Species of a mab, e.g. on Effector Functionality? rel. occurrence of glycans [%] Batch 1 Batch 2 G0F G1F G2F M afucose How to investigate differences in galactose, when mannose and afucose change concomitantly? 2

3 Outline Background / importance of glycosylation of antibodies The in vitro glycoengineering (IVGE) technique Case study on IVGE of an IgG1 Workflow sample preparation Analytical assays applied Results, discussion and summary Summary of IVGE and outlook 3

4 Impact of Fc Glycosylation on Therapeutic Antibodies Sialic acid containing structures suppress ADCC (anti inflammatory); (e.g. Kaneko et al., Science 313, 670-3, 2006) Undesirable glycostructures (potential immunogenicity): α-galactose and NGNA Fully galactosylated structures seem to mediate a better binding to the complement system (CDC; complement-dependent cytotoxicity); (e.g. Kibe et al., J Clin Biochem Nutr 21,57-63,1996) Glycostructures may influence clearance in the plasma (e.g. Newkirk et al., Clin Exp Immunol : ) Glycan structures missing the core fucose can mediate ADCC (antibody-dependent cell-mediated cytotoxicity); (e.g. Okazaki, et al., J Mol Biol 336, , 2004) Drivers for glycan analysis and assessment of glycan structures: Regulatory requirements / guidelines Better understanding of the impact on effector function: ADCC, CDC, clearance 4

5 Glycan Heterogeneity of MABs Current production systems, e.g. CHO cells, exhibit a heterogeneous glycosylation pattern of the expressed therapeutic proteins G0F G1F G2F G0 G1 G2 M5 M6 hm3 hm3f hm3g1f 36-62% 35-43% 9% 7% 3% 1% 7% 1% 2% 8% 5% Homogeneous glycan pattern might be beneficial (analytical reference material or even improved biological activity) Benefit from glycoengineering?

6 Influencing Glycanstructures of mabs Choice of production cell line (CHO, NS0, plant, insect, ) still glycan heterogeneity Cell line engineering (overexpression of glycoenzymes) - time consuming, still glycan heterogeneity Suitable substrates in fermentation process - only limited impact Variation of fermentation parameters (temperature, fermentation time, ) - only limited impact In vitro glycoengineering a new approach?

7 What Does In Vitro Glycoengineering Mean? What is in vitro glycoengineering? Remodeling of glycan structures of therapeutic proteins Applied in vitro, i.e. after the harvest/purification of the therapeutic proteins No genetic engineering of cell lines is necessary / independent from production process Need for enzyme(s) (and activated sugar(s)) which have not been reliably available in the past

8 Recently Developed Glycosyltransferases Galactosylation of IgG4 with GalT1 Sialidation of IgG4 with ST6 rel. content [%] time [h] G0 G1 G2 G0 G1 G2 rel. content [%] time [h] G2+0SA G2+1SA G2+2SA G2+0SA G2+1SA G2+2SA 30μg GalT1/mg IgG4 30μg ST6/300μg IgG4 100% galactosylation after 8 hours 90% sialylation after 6 hours Highly active enzymes as well as activated sugars are available (Roche Custom Biotech) 8

9 Case Study: In Vitro Glycoengineering of IgG1 Five different glycan variants from one single batch by IVGE by use of glycosidases and glycosyltransferases Application of different analytical assays Glycan analysis (verification of IVGE) LCMS peptide mapping (microheterogeneity) Size exclusion chromatography (molecule integrity) FcRn column SPR based: FcyRI, IIa and IIIa FcyR column: FcyRIIa and IIIa ADCC assay M. Thomann, Roche Diagnostics GmbH Bioanalytical Congress, Berlin

10 Sample Preparation Workflow Starting Material Bulk galactosidase & purification GalT1 + UDP-Gal intermediate purification ST6 + CMP-NANA ST6+ CMP-NANA G0 variant (~1day*) G2+2SA variant (~8days*) G2+1SA variant (~8days*) G2 variant (~4days*) * Sample preparation time in days 10

11 Glycopattern After IVGE Sample/Glycan variant name Bulk 1 Bulk 2 G0 G2 G2+1SA G2+2SA G0F G1F rel. occurrence of glycans [%] G2F G2S1F G2S2F M Sum of afucose (w/o M5) Batch variability of glycan levels is smaller than changes introduced by IVGE Batch variability also means changes of multiple species (e.g. galactose, mannose, afucose) Improved evaluation of binding properties of the IgG1 to e.g. FcγRI/II/III and ADCC activity 11

12 Molecular Integrity After IVGE Sample/Glycan variant name Bulk G0 G2 G2+1SA G2+2SA rel. occurrence of microheterogeneity [%] size variant [%] CDR deamid CDR deamid CDR isoasp M255 ox M431 ox Monomer HMW LMW Overall, no major degradation during sample preparation 12

13 Fc Receptor Affinity Column Assays Principle Antibody load to FcRn coated SA beads at ph 6.0 Antibody load to FcγRIIIa coated SA beads at ph 6.0 Antibody elution in a gradient to ph 7.4 Antibody elution in a gradient to ph mab7-aaa WVL:280 nm mau Both HCs fucosylated WVL:280 nm 80 mab7-wt 100%B Absorbance [mau] mab7-yte One or none HC fucosylated Retention Time [min] min T. Schlothauer et al., mabs,

14 Binding Analysis FcRn Column Assay Based on current experience, observed differences in retention time are regarded as not significant no significant impact of galactose or sialic acid on FcRn binding See also: J. Stracke et al, mabs, 2014, A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies 14

15 Binding Analysis FcγRIIa Column Assay Positive impact of galactose Some additional impact from sialic acid 15

16 Binding Analysis FcγRIIIa Column Assay Increased binding of galactosylated variants No impact of sialic acid 16

17 Binding Analysis Fcγ Receptors by SPR FcγRI FcγRIIa FcγRIIIa No substantial impact of Gal and SA on FcγRI binding Increased binding of galactosylated and SA variants in FcγRIIa SPR assay Positive impact of Gal but no additional effect of SA on FcγRIIIa binding 17

18 ADCC Activity (cell based assay) Use of an ADCC assay with increased sensitivity employing engineered natural killer cells (A. Schnueriger et al, MolImmunol, 2011) Positive impact of Gal on ADCC activity Neither positive nor negative contribution of SA to ADCC activity 18

19 Summary Study Results Impact of Fc terminal galactose or sialic acid FcRn binding: No impact FcγRI binding: No impact FcγRIIa binding: Increase in SPR and column assay for Gal and SA FcγRIIIa binding: Increase (SPR & column) for Gal but no impact of SA ADCC activity: Increase for Gal but no impact of SA Publication in preparation M. Thomann, Roche Diagnostics GmbH Bioanalytical Congress, Berlin 2014

20 Discussion Different impact of terminal sugars on biological activity shown in literature possible reasons? Impact of sample preparation Use of different batches: changes in multiple glycan species Fractionation/IVGE (in the past): still low purity of fractions/only small changes in glycan profile appropriate analysis of glycan profile and molecular integrity needed Potency assays used (e.g. PBMCs in ADCC assay) Dependency on investigated molecule (e.g. high level of afucose) Unknown effects, e.g. NANA vs. NGNA, linkage of sialic acid (2,3 vs. 2,6) Further studies needed; investigate your own (new) molecule

21 Summary In Vitro Glycoengineering Besides change of levels of targeted glycan structures (e.g. galactose) levels of other glycan species are not affected (e.g. afucose) Allows for preparation of reference material for analytical assays in medium scale amounts (up to 1g IgG) Allows for unambiguous conclusions about functionality of investigated glycan species Upscaling and integration into established production processes currently tested (e.g. integration into purification process) 21

22 Outlook Benefits of in vitro glycoengineering Issues w/o Glycoengineering Process optimization for glycopattern Process optimization for glycopattern instead of yield Biologic functions of each glycoform Quality by Design Comparability studies, i.e. process change Tech transfer can change glycopattern Manufacturing of biosimilars Benefits using Glycoengineering Less efforts for process optimization Process optimization for yield Biologic functions determined by fewer/one glycoform True implementation of Quality by Design QbD Decreased issues in comparability studies Glycopattern is modified downstream Improved engineering of original glycopattern Improved biopharmaceutical substance 22

23 Acknowledgements Sebastian Malik Tilman Schlothauer & group Petra Rüger & group Alexander Knaupp Cecile Avenal & group Patrick Bulau & group Fredy Schnueriger Roche Diagnostics and Pharma colleagues involved in glycosyltransferase development Bernhard Spensberger Sandra Wöhrl

24 Doing now what patients need next 24