Applications-

Fluorescent Site-Specific Integration
Cell Line Development Platform

Fast to Obtain RCB in 1.5 Months Fast to Know Product Qualities in 1 Month

Applications

Introduction

AlfaCell Enables Various Products to Achieve High Expression

Monoclonal Antibody

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<h4>
	Background
</h4>

	Based on AlfaCell—the world’s first commercialized site-specific integration (SSI) cell
								line development platform—Great Bay Bio has established a rapid and highly efficient
								workflow for cell line development. Using a marketed monoclonal antibody as the model,
								this project highlights GBB’s technical strength in accelerating biologics development
								for the global biopharmaceutical industry.


	 

<h4>
	GBB’s Solution
</h4>

	The AlfaCell platform leverages SSI technology to rapidly develop high-yield and stable
								cell lines. Integrated with high-throughput instrumentation—including FACS, VIPS, and
								Octet—the platform enables efficient clone selection, cell sorting, monoclonality
								analysis, and downstream process development, delivering cell lines ready for
								industrial-scale production.


	Development of high-yield and stable cell lines using site-specific
								integration AlfaCell
								uses homologous recombination to precisely insert target DNA into active and high-titer
								hot spot. This ensures stable and high-yield clones while eliminating variability caused
								by random integration.


	Day 0 – Electroporated plasmid and recombinase into
								the AlfaCell host cell
								line.


	Day 2 – Applied cell selection pressure to eliminate cells
								without correct
								integration.


	Day 4 – 3-day recovery without cell selection pressure;
								non-fluorescent
								single cells sorted into 96-well plates via FACS and cultured for 14 days; VIPS imaging
								tracked monoclonal expansion throughout.


	Day 21 – Measured the titer of monoclonal supernatants via
								Octet; expanded
								TOP24 clones to 24-well plates for scale-up and fed-batch screening.

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	Through fed-batch culture in a shake flask, the TOP3 clones
								achieved
								high-yield.


	Stable integration ensures consistency in key quality attributes (CQAs), including charge
								heterogeneity and glycosylation modifications, across production batches.


	<img src="/static/demo/pop_img4.png" alt="" /> 


	 

<h4>
	Results
</h4>
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	<img src="/static/demo/pop_img5.png" alt="" /> 
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	Stable integration ensures consistency in key quality attributes (CQAs), including charge
								heterogeneity and glycosylation modifications, across production batches.

TCE Drug

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<h4>
	Background
</h4>

	The development of TCE drugs faces multiple technical and biological challenges. Among these, process development is particularly constrained by two major bottlenecks: high mispairing rates and low titer, which are difficult to optimize. While TCE drugs hold great commercial potential,  achieving stable, high-quality manufacturing remains a significant hurdle in their development.


	 

<h4>
	GBB’s Solution
</h4>

	To efficiently address the challenge of low productivity in TCE process development, Great Bay Bio’s AlfaCell platform employs site-specific integration technology, combined with a proprietary single-plasmid quad-cassette design and promoter optimization strategy. This enables the development of high-quality cell lines, significantly improving the titer while reducing mispairing rates, achieving mispairing rates below 20% and the titer consistently above 5 g/L.


	 


	<img src="/uploads/image/20251229/20251229142336_47170.svg" alt="" /> 


	 


	 


	Reducing Competitive Mispairing from Multi-Plasmid Systems 


	Pairing rate directly impacts the success of drug development and commercial feasibility. The single-plasmid quad-cassette design integrates all four genes (H1, H2, L1, L2) into a single vector. By fine-tuning promoter strength, the molar ratio of heavy to light chains approaches 1:1, increasing the correct pairing rate to 90% and enhancing production feasibility.


	 


	Increasing Capacity  for Downstream Process Optimization 


	AlfaCell's site-specific integration technology greatly improved process stability and consistency, minimized expression drift, and allowed better control of key quality attributes like charge variants and glycosylation.


	<img src="/uploads/image/20251229/20251229142447_31923.svg" alt="" /> 


	 

<h4>
	Results
</h4>

	<img src="/uploads/image/20251230/20251230170456_42148.svg" alt="" />


	The AlfaCell platform completed the cell line development for this TCE drug, achieving a titer of 5.6 g/L and a correct pairing rate of 84%.


	 

<h4>
</h4>
<h4>
	TCE Products 
</h4>
<div style="text-align:justify;">
	Great Bay Bio has leveraged its independently developed AI-enabled molecular design platform to screen and obtain CD3 with varying binding affinities, ranging from 0.2 nM to several hundred nanomolar, meeting the diverse requirements of different TCE products.
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	<img src="/uploads/image/20251229/20251229143110_59685.svg" alt="" /> 
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Four-chain BsAb

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<h4>
	Background
</h4>

	A leading global pharma company (Co. A) faced significant technical hurdles during the cell line development phase of their new four-chain BsAb product, including high mispairing rates, high half-antibody levels, and difficulties with multi-plasmid transfection screening, causing project delays.


	 

<h4>
	Pain Points
</h4>

	High mispairing rates 


	Mispaired products such as light-chain and heavy-chain mispairings, increase purification complexity, production costs, and time, making them difficult to commercialize.


	 


	High half-antibody levels 


	The imbalance in heavy and light chain expression often leads to half-antibody production, affecting yield and stability.


	 


	Multi-plasmid transfection screening issues 


	The introduction of multiple plasmids complicates screening and quality control, impacting product development efficiency and production costs.


	 

<h4>
	GBB’s Solution
</h4>

	Great Bay Bio, leveraging the world’s first commercialized site-specific integration cell line development platform, AlfaCell, successfully addressed these issues by integrating multiple expression frames into a single plasmid. Utilizing world-class technologies such as FACS, VIPS, and Octet, we efficiently performed cell line selection, single-cell plating, and monoclonal origin detection, enabling Co. A to quickly establish a high-yield, stable cell line for industrial-scale production.


	 


	 


	<img src="/uploads/image/20251229/20251229144800_34019.svg" alt="" style="width:90%;" /> 


	Reduced mispairing through single-plasmid system 


	By integrating four gene groups (H1, H2, L1, L2) into a single plasmid and optimizing promoter strength, we achieved a 90% pairing rate, significantly reducing mispairing and improving expression efficiency.


	 


	Simplified cell line development 


	AlfaCell reduced the complexity of the process and shortened the development cycle from 4-6 months to just 1.5 months.


	 


	Enhanced process stability & passage batch consistency 


	Stable integration ensures consistency in key quality attributes (CQAs), including charge heterogeneity and glycosylation modifications, across production batches.


	 


	<img src="/uploads/image/20251229/20251229144858_35696.png" alt="" /> 


	 

<h4>
	Results 
</h4>

	<img src="/uploads/image/20251229/20251229144934_49330.svg" alt="" style="width:90%;" />

Nano Bispecific Antibody

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<h4>
	Background
</h4>

	Company B (Co. B) is a biopharmaceutical company focused on innovative immuno-oncology therapies. Using its proprietary next-generation antibody platform, it develops multifunctional antibody drugs. During the development of a nano bispecific antibody product, Co. B (along with several CRO partners) adopted a single open reading frame (ORF) design with random integration for cell line development. However, the resulting expression levels remained too low to support industrial-scale production.


	This nano bispecific antibody project faced a critical deadline: the titer needed to exceed 5 g/L within 3 months, or the project risked termination. 


	 

<h4>
	GBB’s Solution
</h4>

	Great Bay Bio (GBB), leveraging the world’s first commercialized site-specific integration cell line development platform (AlfaCell), utilizes site-specific integration technology for multi-plasmid simultaneous screening. Combined with advanced instruments such as FACS, VIPS, and Octet, we efficiently complete cell line sorting, single-cell plating, monoclonality analysis, as well as analytical and purification process development. This enabled Co. B to rapidly establish high-yield, stable cell lines suitable for industrial production.


	 


	<img src="/uploads/image/20251229/20251229145914_81924.svg" alt="" /> 


	 


	Site-specific integration for high-yield & stable cell line development 


	Through site-specific integration (SSI)-mediated homologous recombination, the target gene is precisely inserted into hot spots that are stable, high-titer, and of high quality. This enables the rapid development of high-yield and stable cell lines, while avoiding the gene expression variability caused by random integration.


	 


	In this project, two sets of codons were optimized based on the molecular characteristics of the protein, and three different plasmids were constructed using distinct plasmid arrangements. Stable cell line development was carried out in parallel using all three plasmids, and monoclonal cell lines were obtained within 1.5 months. Subsequent fed-batch shake flask studies revealed significant differences in expression levels among the three plasmid designs.

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	<img src="/uploads/image/20251229/20251229150031_43933.svg" alt="" /> 


	 


	 


	HTP Cell Sorting, Detection, and Monoclonality Analysis Instruments 


	Stable integration ensures consistency in key quality attributes (CQAs), including charge heterogeneity and glycosylation modifications, across production batches.


	 


	 


	<img src="/uploads/image/20251229/20251229151138_63854.png" alt="" /> 


	 

<h4>
	Results 
</h4>

	<img src="/uploads/image/20251229/20251229150906_94543.svg" alt="" /> 


	Within just 3 months, GBB completed stable cell line development for Co. B's nano bispecific antibody. The top-performing cell line achieved a titer of 15.6 g/L, successfully meeting and exceeding the industrial production target.

Site-Specific Integration Unlocks Step-Change

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<h4>
	Background
</h4>
<div style="text-align:justify;">
	Company A (Co. A), a biotech company focused on developing innovative biologics, used traditional random integration to develop a cell line for one of its core bispecific antibody programs. Despite subsequent process optimization, the titer was limited to 2.2 g/L, which was insufficient to meet the dosage requirements for toxicology and early clinical studies. This bottleneck significantly delayed the IND submission.
</div>

	During the search for third-party collaboration, Co. A leveraged Great Bay Bio’s AlfaCell platform and its site-specific integration technology to increase the cell line titer to 4.0 g/L.


	 


	 


	Key challenges: 


	Low titer from random integration 


	2.2 g/L, with over 70% of integration sites located in transcriptionally silent regions.


	 


	High production cost 


	Estimated over USD 380,000 per batch at 2000L scale.


	 


	Tight clinical timeline 


	Only 9 months remaining before IND filing deadline.


	 


	 

<h4>
	GBB’s Solution
</h4>
<div style="text-align:justify;">
	To overcome this bottleneck, Co. A adopted the AlfaCell platform, leveraging site-specific integration to precisely insert the gene of interest into a highly active and stable genomic hot spot. Integrated with advanced technologies such as FACS, VIPS, and Octet, the platform enabled high-throughput clone selection, monoclonality analysis, and process development—rapidly delivering a high-titer, stable cell line ready for industrial-scale production.
</div>

	 


	Site-specific integration 


	Precisely inserts the gene of interest into a stable, high-titer genomic hot spot, preserving antibody structure and target-binding functionality while minimizing host genome disruption.


	 


	Rapid screening 


	Fluorescence-labeled site-specific streamlines the process by bypassing redundant screening steps.


	 


	<img src="/uploads/image/20251229/20251229152714_15362.svg" alt="" /> 


	 


	 

<h4>
	Results
</h4>
<div style="text-align:justify;">
	After adopting AlfaCell’s site-specific integration technology, the bispecific antibody achieved a titer of 4 g/L within just 1.5 months, with a favorable growth profile.
</div>

	 


	<img src="/uploads/image/20251229/20251229152728_55094.svg" alt="" />

Parallel Cell Line Development Prior to Final PCC Confirmation

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<h4>
	Background
</h4>

	An innovative biopharmaceutical company encountered low expression levels during early transient expression of its BsAb program.


	 


	Traditional drug development typically follows a sequential workflow, which is not only time-consuming but also fraught with significant risks and resource inefficiencies. If the sole selected PCC later demonstrates poor developability, such as low titer, poor stability, or severe aggregation, the project may face major delays or even termination, rendering early-stage investments futile.


	 


	 

<h4>
	GBB’s Solution
</h4>

	To help clients overcome development bottlenecks, Great Bay Bio(GBB) tackles the core challenge by parallel developing high-yield, stable cell lines for multiple potential PCCs during the most critical selection stage. This enables early assessment of expression level and stability, and the resulting stable cell line samples can be used for efficacy studies, minimizing variability caused by differences between cell lines.


	 


	The AlfaDAX platform was used to evaluate multiple candidate molecules, identifying three “dual-excellent” candidates with both strong efficacy and favorable developability. Leveraging the AlfaCell platform, parallel stable cell line development was completed within 1.5 months, with all 14-day shake flask titers exceeding 4 g/L. As a result, the project advanced to the CMC stage significantly faster than the traditional path, while also mitigating the risk of project delay or failure due to cell line issues.


	 

<h4>
	Results
</h4>

	Enhancing PCC selection success rate: balancing efficacy and developability


	At the early stage of molecular design or screening, AlfaDAX leverages its AI-powered platform to evaluate multiple candidate sequences. It not only predicts efficacy-related properties, but also proactively identifies developability risks—such as expression level, solubility, stability, post-translational modifications, aggregation, immunogenicity, and clearance rate. This enables precise selection of molecules with both strong efficacy and favorable developability potential, while filtering out high-risk candidates. As a result, only “dual-excellent” molecules—those that are both effective and easy to develop—enter the parallel development stage, addressing the traditional limitation of focusing solely on efficacy without evaluating developability.


	 


	Seamless integration of PCC and CMC: significantly reducing end-to-end development risk 


	<img src="/uploads/image/20251229/20251229153230_12546.png" alt="" /> 


	 


	The AlfaCell Transient Transfection Kit provides standardized, high-throughput expression assessment for multiple selected candidate molecules, enabling rapid evaluation of product quality. More importantly, the expression vector and host system used in this kit are fully compatible with the subsequent high-yield stable cell line development platform (AlfaCell), achieving seamless transition from transient to stable expression. This unified system minimizes variability in efficacy caused by platform switching. Additionally, one-step plasmid design eliminates the need for time-consuming redesign and re-optimization, further accelerating the development timeline.


	 


	<img src="/uploads/image/20251229/20251229154114_17832.svg" alt="" /> 


	 


	 


	Accelerate project timelines: PCC screening, cell line screening, and process development in parallel 


	Based on the results of AlfaCell transient expression (molecules + optimized plasmids), the AlfaCell platform constructs and screens stable cell lines for multiple molecules in parallel. The platform’s unique "Double-Fast" advantage enables obtaining high-titer and stable cell lines within 1.5 months and rapid prediction of product quality within 1 month, allowing early planning for subsequent process development and capacity expansion.