Module 3.2.S — Drug Substance (TILA-278)

Chemistry, manufacturing and controls document for TILA-278.

Change History

3.2.S Drug Substance — TILA-278

This section describes the drug substance for TILA-278, a recombinant humanized immunoglobulin G1 (IgG1) bispecific monoclonal antibody manufactured by Virtual Biopharma Inc. for the treatment of moderate-to-severe ulcerative colitis (UC). The information is presented in accordance with ICH M4Q(R1) (CTD Quality), ICH Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products), ICH Q5A(R2), Q5B, Q5C, Q5D, and ICH Q11. The drug product (a sterile subcutaneous [SC] solution in a single-use prefilled syringe/autoinjector) is described in Module 3.2.P.

Data are reported for the current commercial manufacturing process and for representative development, toxicology, and clinical lots, including the lots used in the pivotal Phase 2b induction study TILA278-201. Numerical entries carrying the discreet marker "" denote representative values pending reconciliation against the corresponding source datasets at final dossier lock.

3.2.S.1 General Information

3.2.S.1.1 Nomenclature

3.2.S.1.2 Structure

TILA-278 is a monovalent-per-target (1 + 1) bispecific full-length IgG1 antibody assembled from four distinct polypeptide chains:

• Heavy chain A (HC-A): anti-TL1A VH–CH1–hinge–CH2–CH3, bearing the "knob" mutation T366W and the effector-attenuating L234A / L235A / P329G (LALA-PG) substitutions (EU numbering).
• Heavy chain B (HC-B): anti–IL-22RA1 VH–CL–hinge–CH2–CH3, bearing the "hole" mutations T366S / L368A / Y407V plus LALA-PG. HC-B carries the CrossMab CH1↔CL domain crossover to enforce cognate light-chain pairing.
• Light chain A (LC-A): anti-TL1A VL–CL (κ).
• Light chain B (LC-B): anti–IL-22RA1 VL–CH1 (κ, CrossMab-crossed constant domain).

Correct heterodimerization is driven by the complementary KiH interface and stabilized by an engineered inter-CH3 disulfide (S354C / Y349C). The LALA-PG Fc abrogates FcγR and C1q binding — appropriate for a molecule intended to neutralize a cytokine and to agonize a receptor rather than to elicit ADCC/CDC — while the native IgG1 FcRn-binding interface is preserved to support the extended half-life required for SC dosing.

Each heavy chain carries a single conserved N-linked glycosylation site at Asn297 (EU numbering) in the CH2 domain. The intra- and inter-chain disulfide connectivity is that of a canonical human IgG1(κ), confirmed by disulfide mapping (Section 3.2.S.3.1). N-terminal heavy-chain glutamine is largely converted to pyroglutamate; C-terminal lysine is variably processed.

Key structural attributes:

3.2.S.1.3 General Properties

TILA-278 drug substance is a clear to slightly opalescent, colourless to slightly yellow aqueous solution. It is highly soluble in the histidine-based formulation buffer and supports the high protein concentration required for a low-volume SC presentation. Higher-order structure (HOS) has been characterized by far- and near-UV circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC); the thermal unfolding profile shows the expected multi-transition IgG1 behaviour with a CH2 transition onset at ~68°C and a dominant Fab/CH3 transition at ~80–83°C. The molecule exhibits pH- and concentration-dependent conformational and colloidal stability consistent with a well-behaved IgG1, with aggregation and fragmentation being the principal degradation pathways under thermal and low-pH stress (Section 3.2.S.7). Biological activity resides in two independent functions: neutralization of TL1A (arm A) and agonism of IL-22RA1 signalling (arm B), both of which are demonstrable on the same molecule (dual-binding; Section 3.2.S.3.1).

3.2.S.2 Manufacture

3.2.S.2.1 Manufacturer(s)

The drug substance is manufactured, tested, and released by Virtual Biopharma Inc. (or its named contract manufacturing organization). The name, address, and responsibility of each site involved in manufacture, testing, and batch release are provided in the site table. Valid manufacturing authorizations and GMP compliance status are cross-referenced in Module 1.

3.2.S.2.2 Description of Manufacturing Process and Process Controls

TILA-278 is produced by fed-batch mammalian cell culture in a CHO-K1-derived, glutamine-synthetase (GS) selection host cell line, followed by a platform purification train comprising Protein A affinity capture, orthogonal polishing chromatography, dedicated viral inactivation and removal steps, and ultrafiltration/diafiltration (UF/DF) into the final drug-substance buffer. A single production batch is defined as the material derived from one production bioreactor processed through downstream to a single UF/DF pool. Nominal commercial scale is a 2000 L single-use production bioreactor.

Upstream (cell culture):

1. Inoculum expansion / seed train — A single working cell bank (WCB) vial is thawed and expanded through shake-flask and seed-bioreactor stages in chemically defined, animal-component-free medium.
2. Production bioreactor — Fed-batch culture (~14 days) with defined feeds, controlled temperature, pH, dissolved oxygen, and a defined temperature/pH shift to modulate productivity and product-quality attributes (e.g., glycosylation, charge variants). Typical harvest titre ~4–5 g/L.
3. Harvest / clarification — Cells and debris are removed by continuous centrifugation and/or depth filtration followed by 0.2 µm bioburden-reduction filtration to yield clarified harvest.

Downstream (recovery and purification):

4. Protein A affinity chromatography — Product capture with low-pH elution.
5. Low-pH viral inactivation — Hold of the Protein A eluate at pH ~3.5 for ≥60 min, then neutralization (dedicated enveloped-virus inactivation step).
6. Cation-exchange chromatography (CEX, bind–elute) — Reduction of aggregates, charge variants, HCP, and residual Protein A.
7. Anion-exchange chromatography (AEX, flow-through) — Reduction of host-cell DNA, HCP, endotoxin, and adventitious virus (dedicated removal step).
8. Mixed-mode / hydrophobic-interaction polishing — Depletion of high-molecular-weight species and any mispaired/homodimeric product-related species arising from the bispecific format.
9. Viral filtration (nanofiltration, 20 nm) — Dedicated size-based virus removal step.
10. UF/DF and formulation — Concentration and buffer exchange into the histidine-based drug-substance formulation, adjustment to target protein concentration, and addition of surfactant/excipients as specified.
11. Bulk filtration and filling — 0.2 µm filtration and aseptic filling into the drug-substance container-closure system (Section 3.2.S.6), followed by frozen storage at ≤ −40°C.

Process controls. The control strategy is defined per ICH Q8(R2)/Q11 and comprises critical process parameters (CPPs), key process parameters, in-process controls (IPCs) with proven acceptable ranges, and in-process action/acceptance limits for both product-quality and microbiological attributes. Representative IPCs include viable-cell density and viability, titre, bioburden and endotoxin at defined stages, Protein A eluate pH and inactivation hold time/pH, chromatography pool volumes and product concentrations, aggregate/charge-variant IPCs at polishing pools, integrity testing of the virus-retentive filter, and final UF/DF pool protein concentration and pH. Maximum in-process hold times and storage conditions have been established and validated.

3.2.S.2.3 Control of Materials

Cell substrate and cell banking. A two-tiered banking system (master cell bank [MCB] and working cell bank [WCB]) has been established, characterized, and tested in accordance with ICH Q5A(R2), Q5B, and Q5D. Testing includes identity (isoenzyme/genetic), viability, sterility, mycoplasma, in vitro and in vivo adventitious virus assays, transmission electron microscopy for retrovirus-like particles, and bovine/porcine virus testing where relevant. Genetic stability and consistency of the coding sequence, copy number, and expression construct have been demonstrated from the MCB through, and beyond, the limit of in vitro cell age (LIVCA) used for production, per ICH Q5B.

Raw materials. Cell-culture media and feeds are chemically defined and animal-component-free. All raw materials of biological origin (where used) comply with TSE/BSE requirements (EMA/410/01 rev. 3; Ph. Eur. 5.2.8), and compendial materials are controlled to Ph. Eur./USP monographs. Compendial and non-compendial raw-material specifications are provided.

3.2.S.2.4 Controls of Critical Steps and Intermediates

Critical steps and their acceptance criteria (e.g., low-pH viral inactivation pH/time, virus-retentive filtration integrity, Protein A and polishing pool acceptance limits, final UF/DF concentration and pH) are defined with justified limits. In-process pool intermediates and their validated hold conditions/times are listed. No isolated, characterized drug-substance intermediate is stored long-term other than the frozen bulk drug substance.

3.2.S.2.5 Process Validation and/or Evaluation

Process performance qualification (PPQ) was executed on consecutive commercial-scale batches, demonstrating consistent process performance and product quality within predefined acceptance criteria. Impurity clearance (HCP, host-cell DNA, Protein A, media components, selection agent/MSX) was demonstrated with adequate margins.

Viral clearance. Viral clearance was evaluated in a scaled-down, qualified model per ICH Q5A(R2) using a panel of relevant and model viruses across the dedicated and contributory steps (low-pH inactivation, AEX, and nanofiltration; with Protein A contribution assessed). Representative cumulative log reduction values (LRV) are summarized below.

All values. The orthogonal combination of an enveloped-virus inactivation step (low pH) and two size/charge-based removal steps (AEX, nanofiltration) provides robust clearance of both enveloped and non-enveloped viruses; consistent with parvovirus resistance to low pH, no inactivation credit is claimed for MVM/Reo-3 at the low-pH step. Adventitious-agent control for the process and facility is described in Module 3.2.A.2.

3.2.S.2.6 Manufacturing Process Development

The evolution of the manufacturing process from the early clinical/toxicology process to the commercial process is described, together with comparability assessments per ICH Q5E across process changes and scale-up. The toxicology lots and the clinical lots used in TILA278-201 were manufactured by qualified processes; comparability between clinical and commercial material was demonstrated at the level of physicochemical characterization, both potency assays, and product-/process-related impurity profiles. A quality-by-design summary links CPPs to critical quality attributes (CQAs).

3.2.S.3 Characterisation

3.2.S.3.1 Elucidation of Structure and Other Characteristics

TILA-278 has been extensively characterized using orthogonal, state-of-the-art methods per ICH Q6B, with particular attention to the attributes that are specific to a bispecific IgG1 (correct chain pairing, absence of mispaired/homodimeric species, and independent function of both arms).

Primary structure and post-translational modifications.

• Intact and subunit mass (LC-ESI-QTOF MS): the deconvoluted intact mass and the reduced/deglycosylated subunit masses of HC-A, HC-B, LC-A, and LC-B are consistent with the theoretical sequences and confirm the expected heterodimeric assembly and glycoform distribution.
• Peptide mapping (tryptic and Lys-C, RP-UHPLC-UV/MS-MS): >99% sequence coverage confirming both HC and both LC amino-acid sequences and the engineered KiH/LALA-PG/CrossMab substitutions; localization and quantitation of N-terminal pyroglutamate, C-terminal lysine processing, deamidation (Asn), isomerization (Asp), and Met/Trp oxidation hotspots.
• Disulfide mapping and free thiol confirming canonical IgG1(κ) connectivity plus the engineered S354C/Y349C inter-CH3 bond; low free-thiol content.
• Correct chain pairing / bispecificity: LC-MS and orthogonal chromatographic/electrophoretic methods confirm the intended HC-A/HC-B heterodimer with cognate light chains and demonstrate low levels of homodimer, half-antibody, and mispaired species.

Higher-order structure. Far-/near-UV CD and FTIR confirm the expected β-sheet-dominated secondary structure and native tertiary fold; DSC confirms the characteristic multi-transition thermal profile. HOS is comparable between reference standard and process lots.

Glycosylation. Released N-glycan analysis (2-AB labelling, HILIC-UHPLC-FLR with LC-MS confirmation) shows a predominantly core-fucosylated, biantennary complex-type profile dominated by G0F, G1F, and G2F, with minor afucosylated, high-mannose (Man5), and sialylated species; site occupancy at Asn297 is high. Because effector function is engineered out (LALA-PG), afucosylation is not an ADCC-relevant CQA but is monitored for consistency; high-mannose content is monitored for its potential effect on clearance.

Charge and size heterogeneity.

• Charge variants: imaged capillary isoelectric focusing (icIEF), orthogonally confirmed by CEX-HPLC, resolve main, acidic (deamidation, glycation, sialylation), and basic (C-terminal Lys, unprocessed pyroGlu) species.
• Size variants: SE-HPLC quantifies monomer, high-molecular-weight (HMW/aggregate), and low-molecular-weight (LMW/fragment) species; reduced and non-reduced CE-SDS confirm correct assembly and quantify size-based purity and fragments.

Biological characterization — both binding functions.

• Arm A (anti-TL1A antagonist): binding kinetics/affinity to human TL1A by surface plasmon resonance (SPR/Biacore) with a monovalent K_D in the low-nanomolar-to-sub-nanomolar range; TL1A neutralization measured in a cell-based reporter assay in which TILA-278 blocks TL1A/DR3 (TNFRSF25)-driven signalling, expressed as relative potency versus the reference standard.
• Arm B (IL-22RA1 agonist): binding to human IL-22RA1 by SPR; agonist activity measured in a cell-based STAT3 reporter/pSTAT3 assay in IL-22R-responsive cells, in which receptor engagement drives IL-22RA1/IL-10RB–JAK1/TYK2–STAT3 signalling, expressed as relative potency versus the reference standard.
• Dual (simultaneous) binding: a bridging assay demonstrates that a single TILA-278 molecule engages TL1A and IL-22RA1 concurrently, confirming intact bispecific functionality.
• Fc functional confirmation: FcγRIIIa and C1q binding are below the level of the parental wild-type IgG1, confirming effector attenuation; FcRn binding is retained.

3.2.S.3.2 Impurities

Product-related substances and impurities.

• Substances (active): charge and glycoform variants shown to retain both functions.
• Impurities: HMW aggregates; LMW fragments; deamidated/isomerized/oxidized variants; glycation; C-terminal lysine and unprocessed pyroglutamate species; and format-specific species (homodimers, half-antibody, mispaired species). Levels are controlled by the manufacturing process and monitored by SE-HPLC, CE-SDS, icIEF, and mass spectrometry.

Process-related impurities.

Impurity clearance across the purification train has been demonstrated with adequate margins (Section 3.2.S.2.5), and the clinically qualified levels are reflected in the specification (Section 3.2.S.4).

3.2.S.4 Control of Drug Substance

3.2.S.4.1 Specification

The drug-substance specification (ICH Q6B) comprises identity, quantity, purity/impurities, potency (both functions), and safety attributes. Representative acceptance criteria are shown below; individual numerical limits marked "" will be finalized as additional commercial manufacturing data accrue.

3.2.S.4.2 Analytical Procedures

Non-compendial methods (SE-HPLC, CE-SDS reduced/non-reduced, icIEF, peptide-map/binding identity, released N-glycan analysis, both cell-based potency assays, HCP ELISA, host-cell DNA qPCR, Protein A ELISA, UV concentration) are described with sufficient detail to permit execution (system suitability, reference/control standards, calculation of relative potency by parallel-line/four-parameter logistic analysis). Compendial methods (appearance, pH, endotoxin, bioburden) are performed per the referenced Ph. Eur./USP chapters. The two potency assays independently interrogate the two mechanisms of action of the molecule (TL1A antagonism and IL-22RA1 agonism), consistent with the complementary anti-inflammatory and mucosal-healing rationale of the programme.

3.2.S.4.3 Validation of Analytical Procedures

All non-compendial release methods have been validated in accordance with ICH Q2(R2) for the relevant characteristics (specificity, accuracy, precision [repeatability and intermediate precision], linearity, range, and for potency, relative accuracy and dilutional linearity). Compendial methods have been verified for use with TILA-278. Validation summaries and acceptance criteria are provided.

3.2.S.4.4 Batch Analyses

Batch-analysis data are provided for development, toxicology, and clinical drug-substance lots, including the lots used to manufacture drug product for the pivotal Phase 2b study TILA278-201. Representative results are summarized below (all values).

Results across lots are consistent and within the proposed acceptance criteria, supporting process consistency and comparability of the clinically qualified material.

3.2.S.4.5 Justification of Specification

The specification is justified per ICH Q6B on the basis of: (i) the characterization data (Section 3.2.S.3); (ii) the clinically and non-clinically qualified ranges observed in the toxicology lots and the TILA278-201 clinical lots; (iii) manufacturing capability and process consistency (PPQ and batch analyses); (iv) stability behaviour (Section 3.2.S.7); and (v) assay capability. Two orthogonal potency assays are included because both binding functions are essential to the mechanism of action; purity, charge, size, and impurity limits are set to control the CQAs identified in the QbD assessment. Acceptance criteria will be tightened as additional commercial manufacturing experience accrues.

3.2.S.5 Reference Standards or Materials

A two-tiered reference-standard system is used. The primary reference standard (PRS) was established from a well-characterized, representative clinical-quality lot and fully characterized by the orthogonal panel in Section 3.2.S.3. Working reference standards (WRS) are qualified against the PRS using a predefined protocol (identity, potency for both functions, purity, and charge/size profile) and used for routine release and stability potency determinations. The qualification and requalification protocols, storage conditions (≤ −70°C), and traceability of the current standards to the PRS are documented. The reference standard is used to report both potency assays as relative potency.

3.2.S.6 Container Closure System

The drug substance is filled into sterile, single-use bioprocess containers (fluoropolymer/ethylene-vinyl-acetate multilayer bags with appropriate tubing/connectors, or PETG bottles) and stored frozen at ≤ −40°C. Product-contact materials comply with the relevant compendial requirements for plastic containers for pharmaceutical use (Ph. Eur. 3.1.x; USP <87>/<88> biological reactivity, Class VI) and food-contact regulations. Container-closure integrity and suitability (protection, compatibility, and safety) have been demonstrated, and an extractables/leachables assessment appropriate to a frozen protein drug substance has been performed. The maximum frozen storage duration and permitted freeze/thaw cycles are defined and supported by stability data (Section 3.2.S.7).

3.2.S.7 Stability

3.2.S.7.1 Stability Summary and Conclusions

Stability of the drug substance has been evaluated under ICH Q5C and Q1A(R2) conditions using stability-indicating methods (SE-HPLC, CE-SDS, icIEF, both potency assays, appearance, pH, protein concentration, and sub-visible particles). Studies include long-term storage at the intended condition (≤ −40°C), accelerated (5 ± 3°C), and stressed/thermal (25 ± 2°C and 40 ± 2°C) conditions, together with freeze–thaw cycling and forced-degradation and photostability (ICH Q1B) studies to demonstrate method specificity and to characterize degradation pathways.

Under the intended frozen storage condition, TILA-278 drug substance is stable, with all attributes remaining within acceptance criteria for the full duration tested. The principal degradation pathways observed under accelerated/stressed conditions are aggregation (increase in SEC HMW), fragmentation (CE-SDS), and charge-variant shifts (increase in acidic species, reflecting deamidation), accompanied by gradual loss of relative potency at thermal-stress conditions; both potency assays track molecular integrity. No unexpected degradants were observed. Freeze–thaw cycling within the defined number of cycles produced no meaningful change.

Based on the available long-term data at ≤ −40°C and supported by accelerated/stressed data, a drug-substance shelf life of 36 months at ≤ −40°C is proposed.

3.2.S.7.2 Post-approval Stability Protocol and Stability Commitments

Virtual Biopharma Inc. commits to: (i) continue long-term stability testing of the primary batches through the full proposed shelf life; and (ii) place the first commercial-scale batches on the post-approval long-term stability protocol, testing at the defined intervals using the methods and acceptance criteria above. Any confirmed out-of-specification or significant adverse trend will be reported to the health authority per regional requirements.

3.2.S.7.3 Stability Data

Detailed stability data tables for all primary and supporting batches (including the toxicology and TILA278-201 clinical lots) are provided, with tabulated and graphical presentation of each attribute versus time and statistical evaluation of the long-term data supporting the proposed shelf life.

Cross-references: adventitious-agent safety (3.2.A.2); manufacturing facilities (3.2.A.1); excipients and drug-product formulation, container-closure, and stability for the SC prefilled syringe/autoinjector (3.2.P); regional information (3.2.R).