At Elise Biopharma, we recognize that the sugar chains attached to your therapeutic proteins—known as glycans—are far more than decorations. They govern half-life, efficacy, immunogenicity, and even safety. Our Glycoengineering & Glycoform Optimization services transform host cells into precision machines, sculpting glycan profiles to your exact specifications. Whether you’re developing next-generation antibodies, enzyme replacements, or Fc-fusion decoys, our integrated glycosylation engineering platform ensures optimal glycoform distributions, consistent batch-to-batch quality, and a fast track to clinical success.

Why Glycoengineering Matters

1. Extend Serum Half-Life
   • Sialylation and terminal galactose residues engage with hepatic lectin receptors to delay clearance.
   • Optimized Fc glycoforms exploit FcRn recycling for multi-week dosing intervals.
2. Tune Effector Functions
   • Afucosylation enhances ADCC by increasing affinity to FcγRIIIa on NK cells.
   • Controlled bisecting N-acetylglucosamine (GlcNAc) modulates CDC and ADCP.
3. Reduce Immunogenicity
   • Human-like glycan patterns minimize anti-drug antibody (ADA) responses.
   • Eliminate non-human structures (α-Gal, Neu5Gc) that trigger hypersensitivity.
4. Improve Stability & Solubility
   • Certain glycoforms shield hydrophobic patches, reducing aggregation.
   • Glycan–protein interactions can stabilize tertiary structure under stress.

Our Glycoengineering CDMO Platform

Elise Biopharma’s proprietary platform integrates molecular design, cell line engineering, process development, and advanced analytics into a seamless glycoform optimization workflow:

Module	Capabilities
Host Cell Engineering	CHO, HEK293, Pichia pastoris with targeted knock-ins/knock-outs (GNTI, FUT8, ST3GAL, B4GALT)
Vector & Expression	Glyco-optimized expression cassettes, signal peptide tuning, promoter selection for balanced expression
Upstream Process Design	Media supplementation with Mn²⁺, uridine; feed strategy for glycan precursors; temperature and pH modulation
Downstream Purification	Lectin affinity chromatography, mixed-mode resins for glycoform fractionation, size-exclusion polishing
Analytical Characterization	LC-MS/MS glycopeptide mapping, HILIC-FLD glycan profiling, CE-SDS for charge variants
Regulatory Support	CMC documentation for glycan critical quality attributes (CQAs), comparability protocols, IND/BLA filing support

Host Cell Engineering & Strain Selection

3.1 Choosing the Right Host

• CHO Cells
  • Gold standard for human-like N-glycans; robust growth and regulatory familiarity.
• HEK293 Cells
  • Superior sialylation machinery; valuable for highly sialylated glycoforms.
• P. pastoris (Glycoengineered Strains)
  • Rapid growth and simple media; custom strains programmed for human-type glycosylation.

3.2 Genetic Modifications

• Knock-Outs
  • GNTI-KO: Produces high-mannose glycoforms for rapid clearance models or specific enzyme products.
  • FUT8-KO: Eliminates core fucose for enhanced ADCC.
• Knock-Ins
  • ST3GAL1/4, B4GALT1/2: Increase terminal sialylation and galactosylation.
  • Human α-2,6-sialyltransferase to achieve human-specific sialic acid linkages.

3.3 Single-Cell Cloning & Screening

Using FACS and automated imaging, we isolate monoclonal cell lines with optimal growth, productivity, and glycoform consistency. Stability studies confirm > 90% desired glycoform retention over 60+ population doublings.

Upstream Process Development for Glycoform Control

4.1 Media Design & Feed Strategies

• Media Supplements:
  • Manganese (Mn²⁺): Cofactor for galactosyltransferases.
  • Uridine & Galactose: Precursor sugars for glycan extension.
• Feed Timing & Composition:
  • Bolus vs. continuous feed to maintain steady‐state sugar levels.
  • Custom feed blends to balance growth vs. glycosylation pathways.

4.2 Bioreactor Parameter Optimization

• pH Shifts:
  • Slight alkaline shift (pH 7.2–7.4) favors galactosylation.
• Temperature Reduction:
  • Lowering from 37 °C to 30–32 °C post-induction extends glycosyltransferase half-life.
• Dissolved Oxygen (DO):
  • High DO (> 40% saturation) ensures energy for glycan biosynthesis.

4.3 High-Throughput DoE Screening

Using micro-bioreactors (AMBR®), we run factorial designs to pinpoint critical process parameters (CPPs) that drive glycoform distributions. Typical screening matrix:

Variable	Levels
Temperature (°C)	30, 32, 34
pH Setpoint	7.0, 7.2, 7.4
Mn²⁺ (µM)	10, 20, 40
Feed Rate (mL/L/h)	0.5, 1.0, 1.5

Downstream Purification & Glycoform Fractionation

5.1 Lectin Affinity Chromatography

• Concanavalin A (ConA): Captures high-mannose and hybrid glycans.
• Aleuria aurantia lectin (AAL): Binds fucosylated glycoforms for targeted removal.

5.2 Mixed-Mode & IEX Polishing

• Mixed-Mode Resins: Combine ionic and hydrophobic interactions to resolve closely related glycoforms.
• Cation/Anion Exchange: Separate charge variants arising from sialylation differences.

5.3 Size-Exclusion & HIC

• SEC: Final polishing to remove high-molecular-weight aggregates that can co-elute with certain glycoforms.
• HIC: Exploit subtle hydrophobicity changes introduced by glycan modifications.

Analytical Characterization & Quality Control

Assay	Purpose
LC-MS/MS Glycopeptide Mapping	Site-specific glycan composition
HILIC-FLD Glycan Profiling	Quantitative glycan distribution (2AA labeling)
CE-SDS Charge Variant Analysis	Detects sialylation and deamidation variants
1D/2D-SDS-PAGE & Western Blot	Assess molecular weight shifts
SPR/BLI FcRn Binding	Functional half-life correlate
Cell-Based Fc Effector Assays	ADCC, CDC, and ADCP potency
Stability Studies	Real-time & accelerated glycoform integrity
Endotoxin & Host-Cell Protein (HCP)	Ensure safety and purity

Each batch is benchmarked against pre-defined glycan critical quality attributes (CQAs) to guarantee consistency and regulatory compliance.

Regulatory & CMC Support

• Glycan CQA Definition: Establish glycoform ranges critical for safety and efficacy.
• Comparability Protocols: Demonstrate equivalence between R&D and GMP lots via head-to-head analytical studies.
• IND/BLA Filings: Comprehensive CMC documentation, including glycosylation pathways, CPP tables, and analytical methods validation.
• Global Compliance: Alignment with FDA, EMA, and ICH Q6B guidelines on glycan characterization.

Case Study: Afucosylated Antibody Enhancement

Challenge: A mid-stage oncology biotech needed an afucosylated monoclonal antibody to boost ADCC.
Solution Workflow:

1. FUT8 Knock-Out in CHO-S host via CRISPR/Cas9.
2. Screening & Cloning: 200 single clones evaluated; top line produced 4 g/L with < 5% fucosylation.
3. Process Development: Optimized feed strategy with Mn²⁺ depletion post-peak to prevent residual fucosylation.
4. Analytics: Achieved > 95% afucosylation confirmed by HILIC-FLD.
5. GMP Manufacturing: Two 1,000 L runs delivered clinical-grade drug substance with consistent glycoform profiles.

Outcome: IND clearance in 8 months; first-in-human trial initiated on schedule.

Workflow: From Concept to Clinic

1. Discovery & Feasibility (4–6 weeks)
   • In silico glycosylation modeling and target glycoform specification.
   • Small-scale expression screen and preliminary glycan analysis.
2. Cell Line Generation (8–12 weeks)
   • Host engineering, vector design, transfection, and single-cell cloning.
   • Productivity and glycoform screening in multiwell plates.
3. Process Definition (6–8 weeks)
   • Micro-bioreactor DoE for CPP identification.
   • Scale-up to bench-top bioreactors with optimized feed and control strategies.
4. GMP Manufacturing (12–16 weeks)
   • Technology transfer, facility qualification, and GMP campaign scheduling.
   • Bulk drug substance production, downstream purification, and release testing.
5. Regulatory Filing & Support (Concurrent)
   • CMC dossier preparation, glycan CQA justifications, and assay validations.
   • IND/BLA submission assistance and health authority interactions.
6. Post-Launch Support
   • Continuous process verification (CPV), comparability for lifecycle management, and process improvements.

(FAQs)

1. What is the typical timeline for glycoengineering CDMO projects?
   • From feasibility to IND-enabling GMP batches: ~6–9 months, depending on complexity.
2. Can you customize glycoforms beyond common Fc glycan patterns?
   • Yes. We engineer host pathways to produce hybrid, high-mannose, or novel glycoarchitectures on demand.
3. How do you ensure batch-to-batch consistency?
   • Rigorous CPP control, real-time analytics, and CPV frameworks guarantee < 5% glycoform variability.
4. Are your processes cGMP-compliant?
   • Absolutely. Our state-of-the-art facility follows FDA 21 CFR Part 211, EU GMP, and ICH Q6B guidelines.
5. What analytical capabilities do you offer for glycan profiling?
   • LC-MS/MS, HILIC-FLD, CE-SDS, SPR, cell-based assays, and more—as detailed above.

Ready to Elevate Your Glycoform Profiles?

Partner with Elise Biopharma—the premier glycoengineering CDMO—to harness the full power of glycosylation engineering.