Q1: What is AlfaCell?

AlfaCell is a fluorescence-based Site-Specific Integration (SSI) cell line development platform independently developed by Great Bay Bio, focused on the rapid development of high-expression, high-stability, and high-quality production cell lines for biologics.

AlfaCell combines SSI technology, fluorescence visual screening technology, and high-throughput analytical technologies such as FACS, VIPS, and Octet to precisely integrate the target gene into a validated, highly active expression region and rapidly screen for stable, high-yield monoclonal cell lines.

Compared with traditional random-integration cell line development, AlfaCell improves development efficiency, reduces expression-variability risk, and accelerates biologics CMC development.

Q2: What is Site-Specific Integration (SSI) cell line development?

SSI is a genetic engineering technology that uses site-specific recombinases to precisely insert the gene of interest into a specific locus of the host cell chromosome. It enables the cell line to achieve: better expression stability; higher batch-to-batch consistency; and lower CMC development risk.

Q3: On which host cell is AlfaCell developed? Does it support CHO cell line development?

AlfaCell is an SSI cell line development platform built on the CHO-K1 (Chinese Hamster Ovary-K1) cell line, dedicated to developing high-expression, high-stability production cell lines for complex biologics such as antibodies, bispecific antibodies, and fusion proteins.

Leveraging the mature industrial foundation of CHO-K1, AlfaCell combines SSI technology, fluorescence visual screening, and high-throughput monoclonal screening to achieve precise target-gene integration and rapid screening of high-potential cell lines.

As one of the most widely used host cells in global commercial biologics manufacturing, CHO-K1 offers: high process maturity; rich scale-up experience; high regulatory acceptance; and applicability to diverse recombinant protein expression.

Based on the CHO-K1 system, AlfaCell can support cell line development for multiple biologic molecule types — including mAbs, bispecific antibodies, multispecific antibodies, ADC-related antibodies, and Fc-fusion proteins — and meets the requirements of downstream CMC development and commercial manufacturing.

Q4: How does AlfaCell achieve precise gene integration?

Based on the CHO-K1 host cell system, AlfaCell employs site-specific recombinase-mediated technology to precisely insert the target gene into a pre-designed genomic locus, achieving stable integration and sustained expression of the exogenous gene.

Compared with traditional random integration, by controlling the insertion position of the target gene, AlfaCell reduces position-effect and expression-variability risks caused by random integration-site heterogeneity, improving the stability and predictability of cell line development.

After precise gene integration, AlfaCell combines fluorescence visual screening, single-cell sorting, and expression analysis to rapidly screen for high-expression, high-stability monoclonal production cell lines.

Q5: How does AlfaCell select the target integration site?

The core of SSI cell line development lies in selecting genomic integration sites with good expression potential and long-term stability, and precisely directing the target gene into that site.

Based on the CHO-K1 host system, AlfaCell systematically evaluates genomic regions and uses AI technology and high-throughput automated screening to identify and validate highly active expression regions as target integration sites with the following characteristics:

· Open chromatin suitable for accommodating long exogenous gene fragments;

· Ability to support long-term genetic stable expression;

· Reduced risk of gene silencing and expression fluctuation.

After the target site is determined, AlfaCell uses site-specific recombinase-mediated technology to precisely insert the expression cassette into the pre-validated high-expression genomic locus, avoiding the uncontrolled insertion-position problem of traditional random integration, which can adversely affect host cell physiology.

Subsequently, fluorescence visual screening, single-cell sorting, and expression analysis are used to perform high-throughput screening of the integrated cells, yielding high-expression, high-stability candidate monoclonal cell lines.

Q6: How does AlfaCell verify whether the target gene is precisely integrated?

AlfaCell confirms candidate cell lines through a multi-dimensional analysis strategy, including:

· Residual fluorescent-marker analysis;

· Gene-integration analysis;

· Monoclonal-origin confirmation;

· Exogenous-gene expression detection;

· Protein-expression-level analysis;

· Stability evaluation.

Combined with fluorescence screening and high-throughput detection technologies, candidate cell lines with development potential can be rapidly identified.

Q7: How does AlfaCell ensure monoclonality?

AlfaCell combines VIPS single-cell sorting technology, monoclonal-origin analysis, and subsequent verification testing to ensure candidate cell lines originate from a single cell clone.

Screening and analysis at the single-cell level reduce the risk of mixed clones and provide assurance for subsequent stable expression and production consistency.

Q8: What are the advantages of AlfaCell's fluorescence screening technology?

AlfaCell combines fluorescence technology with SSI technology, enabling rapid observation of target-gene integration and expression status.

Combined with:

· FACS flow sorting;

· VIPS single-cell imaging sorting;

· Octet protein-expression detection;

it can rapidly identify:

· High-expression clones;

· Monoclonal-origin cells;

· Candidate cell lines with industrial production potential.

This approach reduces the blindness of traditional screening workflows and improves development efficiency.

Q9: Why does SSI improve the predictability of cell line development compared with random integration?

During random integration, the exogenous gene may randomly enter different genomic regions, and different insertion positions may lead to:

· High host-cell physiological uncertainty;

· Expression-level variation;

· Gene silencing;

· Declining cell line stability.

By controlling the target-gene insertion position, SSI standardizes the development process across different projects and improves the predictability of cell line performance.

For commercial biologics manufacturing, a stable and controllable integration method helps reduce downstream CMC development risk and improve process scale-up and production consistency.

Q10: What is the difference between AlfaCell SSI cell lines and traditional random-integration CHO cell lines?

Traditional random-integration CHO cell line development typically requires screening high-expression clones from a large pool of randomly integrated clones, with problems such as long timelines, high screening costs, and uncertain outcomes.

AlfaCell adopts an SSI strategy, directing the target gene into a stable, high-expression region and combining fluorescence-assisted screening to rapidly obtain high-potential clones. Key advantages include:

· High controllability of host cell physiology;

· Genomic stability;

· More controllable integration sites;

· More stable expression performance;

· Reduced large-scale blind screening;

· Shortened cell line development cycle.

Q11: During cell line development, why is AlfaCell's development cycle shorter than traditional CHO cell line development?

Traditional CHO cell line development usually relies on random integration and extensive clone screening, requiring the search for high-expression clones among a large candidate pool, resulting in a longer development cycle.

Based on the CHO-K1 SSI cell line platform, AlfaCell uses site-specific recombinase-mediated precise gene integration to direct the target gene into predefined sites, and combines fluorescence visual screening, single-cell sorting, and high-throughput analysis to improve candidate-clone screening efficiency.

By reducing ineffective work in random screening, AlfaCell can significantly shorten the cell line development cycle; public cases show the fastest RCB (Research Cell Bank) construction in about 1.5 months.

Q12: Why are SSI cell lines more suitable for industrial biologics manufacturing?

Industrial manufacturing requires cell lines with long-term stable expression capability and batch-to-batch consistency.

By reducing the position effect caused by random insertion, SSI gives cell lines better stability and predictability during:

· Host-cell physiological control;

· Genomic stability;

· Long-term passage;

· Process scale-up;

· Production batch changeover.

Therefore, SSI has become an important technology direction for the development of next-generation high-quality production cell lines.

Q13: Can cell lines developed with AlfaCell undergo long-term stability studies?

Yes.

Based on SSI technology, by controlling the target-gene insertion position, AlfaCell reduces the expression-fluctuation and gene-silencing risks caused by insertion-site differences in traditional random integration, improving the predictability of long-term stable expression from the source.

During cell line development, AlfaCell performs long-term stability assessment of candidate cell lines through stability evaluation, expression-level analysis, and continuous passage observation.

By monitoring the cell line during continuous culture — including:

· Expression maintenance capability;

· Cell growth characteristics;

· Product quality attribute changes;

AlfaCell evaluates the cell line's long-term production potential, providing data support for downstream CMC development, process scale-up, and commercial manufacturing.

Q14: Do cell lines developed with AlfaCell require long-term drug pressure?

No.

Compared with other cell line construction methods (which require long-term drug selective pressure to maintain the stability of the integrated exogenous gene), AlfaCell-built cell lines are selected for the integrated gene using only 48 hours of hygromycin pressure during the transfection stage. After sorting into plates and expansion to the RCB stage, no further drug pressure is needed. The AlfaCell-derived RCB can be used directly to establish the Master Cell Bank (MCB).

Q15: Can AlfaCell reduce CMC development risk?

Yes.

Through SSI, stable-expression screening, and early quality analysis, AlfaCell can help customers:

· Assess expression potential earlier;

· Reduce cell-line instability risk;

· Identify development issues sooner.

Q1: What types of biologic molecules does AlfaCell support?

AlfaCell supports multiple biologic molecule types, including:

· Monoclonal antibodies (mAbs);

· Bispecific antibodies;

· Multispecific antibodies;

· ADC-related antibodies;

· Fc-fusion proteins;

· Other recombinant proteins.

Public cases show AlfaCell has been applied to cell line development for molecules of varying complexity.

Q2: Does AlfaCell support complex molecule development such as bispecific and multispecific antibodies?

Yes.

Complex molecules such as bispecific and multispecific antibodies typically face challenges including:

· Difficulty in coordinating multi-chain expression;

· Insufficient chain-pairing efficiency;

· Complex product quality control.

AlfaCell has completed multiple complex-antibody projects, e.g., both bispecific and multispecific antibody projects achieved stable expression. In public cases, bispecific/multispecific projects reached an average titer of 5.6 g/L, and complex-structure bispecific projects reached fed-batch titers above 6.4 g/L.

Q3: Does AlfaCell support biosimilar development?

Yes.

AlfaCell can be applied to stable cell line development for biosimilar projects, establishing high-expression production cell lines through SSI and high-throughput screening. The platform is suitable for the R&D needs of various antibody and recombinant-protein biosimilars.

Q1: Has AlfaCell been validated by real customer projects?

Yes.

AlfaCell has completed multiple customer projects covering:

· mAbs;

· Bispecific antibodies;

· Multispecific antibodies;

· Fc-fusion proteins;

· Complex-structure proteins.

Public case data show that the 12 most recent customer projects spanning different product types achieved an average titer exceeding 6.9 g/L.

Q2: What are the expression levels of AlfaCell customer projects?

Expression levels of different molecule types are affected by molecular structure, host cell, and culture process. According to public cases:

· mAb projects: average titer approx. 8.1 g/L;

· Bispecific/multispecific projects: average titer approx. 5.6 g/L;

· Fc-fusion protein projects: average titer approx. 10.0 g/L.

Q3: Does AlfaCell have successful cases with complex molecules?

Yes.

Public cases show:

· A four-chain bispecific antibody project completed cell line development in about 3 months at a titer of 8.3 g/L;

· A nanobody-derived bispecific antibody project reached a titer of 15.6 g/L;

· A bispecific antibody project obtained a stable cell line in as fast as about 1.5 months at a titer above 4 g/L.

These cases demonstrate AlfaCell's ability to support structurally complex molecule development.

Q4: Have AlfaCell SSI cell lines been validated in clinical-stage projects?

Public cases show that a customer innovative-drug project using AlfaCell-developed cell lines has achieved out-licensing and advanced to Phase II clinical stage, while also receiving FDA Fast Track Designation.

This case indicates that AlfaCell-developed cell lines can meet the quality and stability requirements of innovative biologic projects throughout early R&D, cell line development, and clinical-stage advancement.

Because innovative-drug projects involve customer trade secrets, specific customer names, product information, and detailed development data are disclosed only upon customer authorization.

Q5: Does AlfaCell support IND filing?

Yes.

AlfaCell has completed FDA Drug Master File (DMF) filing, DMF number 038272. When customers use AlfaCell-developed cell lines for new-drug registration, relevant DMF information can be referenced per regulatory requirements to support IND filing.

Q6: Does AlfaCell own intellectual property?

Yes.

AlfaCell's integration hotspot within our host cell line is protected by patents filed in multiple countries. An authoritative IP law firm issued a Freedom to Operate (FTO) analysis, confirming that this integration hotspot and its related elements are freely implementable.

Q1: What cooperation models does AlfaCell offer?

AlfaCell provides flexible cooperation models to meet the cell line development and platform-capability needs of biopharma enterprises at different stages, mainly including:

1. Cell Line Development Service

· Customers provide target molecule information; Great Bay Bio completes SSI cell line development on the AlfaCell platform, including: target gene construction and integration; high-expression clone screening; monoclonal cell line establishment; expression and quality analysis; Research Cell Bank (RCB) construction; and delivery of relevant technical documentation.

This model suits Biotech and pharma enterprises that want to quickly obtain high-quality production cell lines but do not plan to build their own cell line development platform.

2. AlfaCell Platform Licensing

· For enterprises with in-house R&D and CMC teams seeking to build autonomous cell line development capability, Great Bay Bio can provide AlfaCell platform licensing cooperation, giving partners access to AlfaCell-related technical capabilities for in-house cell line development process build-out and accelerating their biologics R&D and manufacturing capability layout.

Suitable for: large pharma enterprises; Biotech companies with in-house cell line development teams; enterprises wishing to build long-term autonomous CMC capability; and CDMO enterprises.

Great Bay Bio can provide flexible solutions ranging from project services to platform cooperation based on the customer's R&D stage, technical needs, and business plan, helping customers accelerate the transition from candidate molecule to commercial manufacturing.

Q2: What is the difference between the cell line development service and the technology licensing model?

The two models differ mainly in how technical capability is deployed:

· Cell Line Development Service model: Great Bay Bio completes cell line development and the customer receives the project deliverables — suitable for enterprises wanting to advance projects quickly;

· Technology Platform Licensing model: the customer receives authorization to AlfaCell's technical capabilities and can conduct cell line development within its own R&D system — suitable for enterprises building long-term in-house capability.

Q3: Does AlfaCell provide cell line development outsourcing services?

Yes. Great Bay Bio can provide customized cell line development services on the AlfaCell platform, helping customers complete the development workflow from target molecule to stable production cell line. To date, Great Bay Bio's cell line development outsourcing service has served dozens of domestic and international pharma companies and CDMOs.

Q4: Does AlfaCell support platform technology transfer / licensing?

Yes. For enterprises wishing to build autonomous cell line development capability, Great Bay Bio can provide an AlfaCell platform licensing cooperation plan to help customers construct in-house SSI cell line development capability.

Q5: What is the AlfaCell cell line development workflow?

A typical development workflow includes:

· Project requirement analysis;

· Target gene design and construction;

· SSI;

· Fluorescence-assisted screening;

· Monoclonal establishment;

· Expression and quality analysis;

· RCB construction;

· Technical delivery.

Q6: What are the AlfaCell project deliverables?

Depending on the cooperation plan, deliverables include:

· Stably expressing cell line;

· RCB;

· Monoclonal-origin analysis data;

· Expression detection data;

· Product quality analysis data;

· Technical report.

Q7: Does AlfaCell support customer-designated CDMO manufacturing?

Yes. Great Bay Bio can provide cell line development services and connect with the customer's designated CDMO or manufacturing partner through technology transfer.

AlfaCell considers downstream scale-up needs during cell line construction and can support the transition from R&D to commercial manufacturing. Projects using AlfaCell cell lines have already entered Phase II clinical stage and completed scale-up above 500 L.

Q8: How are customer project IP rights protected?

Customer project items — including:

· Molecule information;

· Cell lines;

· Experimental data;

· Technical documentation;

— and the above core trade secrets are managed per the confidentiality and commercial-contract clauses.

Q1: Why choose AlfaCell for cell line development?

Main reasons to choose AlfaCell include:

· SSI technology improves expression stability and avoids the instability of random integration;

· Fluorescence-assisted screening improves development efficiency and reduces monoclonal workload;

· Supports multiple complex biologic molecules;

· Rapid generation of a stable cell line within 1.5 months;

· Supports downstream CMC and commercialization development.

Q2: How long does it take to obtain a stable CHO cell line with AlfaCell?

According to public cases, AlfaCell can complete RCB construction in as fast as about 1.5 months. The specific timeline depends on:

· Molecule complexity;

· Analytical requirements;

· Project deliverable scope.

Q3: Is AlfaCell suitable for Biotech companies?

Yes.

For Biotech enterprises lacking in-house cell line development capability and wanting to rapidly advance CMC development, AlfaCell can provide:

· Professional cell line development capability;

· Rapid project delivery;

· Downstream CDMO connection support.

Q4: How can I tell whether my molecule is suitable for AlfaCell development?

Customers can provide:

· Molecule type;

· Gene sequence or related information;

· Target titer;

· Development stage;

· Project timeline.

Great Bay Bio will conduct a technical assessment under a Non-Disclosure Agreement (NDA) framework and formulate a suitable cell line development plan.