Strategy for Derivation and Optimization of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production

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BioPharm International, BioPharm International, May 2024, Volume 37, Issue 5
Pages: 11-16

This commentary summarizes the derivation of clonal HEK293 suspension cell lines, selection of clones for rAAV production, and design of experiments-based optimization strategies for characterization of one clonal isolate for high yield rAAV manufacture. Data shown provides proof of concept for the success of this strategy, its applicability for manufacturing different rAAV serotypes and lays the foundation of further clonal cell line characterization for scale up of production.

Industrial manufacture of biotherapeutic drugs and recombinant therapeutic proteins relies predominantly on mammalian cell lines, notably Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293. The HEK293 cell line was originally derived in 1977 by transforming primary human embryonic kidney cells with sheared adenovirus DNA (1). The original cell line has been used to derive several cell lineages over the intervening years (2,3). HEK293 is preferentially used for the manufacture of recombinant adeno-associated viruses (rAAV). Seven rAAV-based gene therapy products, beginning with Glybera in 2012 and Luxturna in 2017, have received regulatory approval for administration to human patients (4). Regulatory agencies recommend that mammalian cell lines expressing recombinant reagents used in commercial manufacturing are derived from single progenitor cells (i.e., these cell lines are monoclonal in origin, “…a collection of cells of uniform composition derived from a single tissue or cell”) (5,6,7). The quality of the biotherapeutic reagent produced by these cells is at the core of this recommendation; any variability in the quality is likely to have an impact not only on the efficacy of the product but may also impact the response in clinical trials. One of the major perceived advantages in using clonal cell lines in manufacturing biotherapeutic reagents is that it facilitates better control of the manufacturing process by reducing background genetic heterogeneity of the cells. This results in more rigorous consistency in the quality of the resultant biotherapeutic reagents.

Historically, clonal cell lines have been derived by the limiting dilution method using cell seeding into 96-well plates to isolate monoclonal cell lines. This is an arduous procedure that, because of its complexity, can be difficult to track and document to the satisfaction of regulatory agencies. Poisson distribution statistics are used to model the likely probability of clonality of the resulting cell lines (8). Typically, two rounds of limiting dilution are required to confirm clonality of the resulting cell lines, a procedural adjustment that is required for regulatory acceptance of clonal origin.

There are several integrated platforms available for cell line development that capture and document what was originally an extremely labor-intensive endeavor. Many of these platforms, include an integrated imaging system which is fundamental to the initial seeding of single cells into 96-well or nanowell plates. The systems enable subsequent tracking and documentation of clonal outgrowth over time and can be used to compile initial data on clonal viability and density. These platforms provide image-based assurance of clonality and facilitate regulatory submissions for filing investigational new drug application (INDs).

Generation of a suspension-adapted, serum-free clonal HEK293 cell line

A high yielding, robust suspension platform for AAV manufacturing (Andelyn Biosciences) demonstrates scalability from 125 mL shaker flasks to stirred tank reactor (STR) 2000L bioreactors using a commercial HEK293 cell line (9–16). A design-of-experiments (DoE) approach was implemented to formulate a model for optimized AAV production and purification. A suspension-adapted HEK293 clonal cell line has been derived and developed to complement this suspension platform for AAV manufacturing. This report highlights critical aspects of the early development work on the clonal suspension cell line.

An adherent HEK293 Good Manufacturing Practice (GMP)-grade Master Cell Bank (MCB) (Andelyn Biosciences) was used as the starting material to derive a series of clonal suspension-adapted HEK293 cell lines. The GMP-MCB was chosen as it had been already subjected to rigorous testing upon its inception for qualification and release. Initially, the adherent HEK293 cell line was adapted to growth in a series of decreasing serum concentrations, from 10% to 5% to 2% to 1% (v/v). Cell lines were then switched to growth in serum-free media, concurrent with growth under suspension conditions. At this point, they were designated as suspension-adapted, serum-free (SASF) cell lines. A total of 15 non-clonal (parental) cell lines were derived from this process.

Isolation of single cell clones from SASF cell lines

Preliminary characterization of early- and mid-passage cells suggested that the cell lines were still adapting to their suspension culture conditions. Growth, viability, and lack of clumping were key criteria in evaluating the suitability of parental SASF cell lines for subsequent clonal derivation. Verified in situ plate seeding (VIPS) was conducted with a cell development platform (Solentim VIPS, Advanced Instruments). This instrument is Code of Federal Regulations (CFR) 21 Part 11 compliant and provides Day 0 assurance of cell line clonality. Mid-passage SASF cells at a density of 1.2 E+4 cells/mL were prepared and used to seed 96-well plates with a single cell seeder. Plates were seeded at a final volume of 125 µL suspension media, supplemented with 10% (v/v) conditioned media (InstiGRO HEK, Advanced Instruments).

A representative plate map from the seeding event outcomes is shown in Figure 1. The data generated provide an accurate record of the seeding events as they occurred in real time and thus provide assurance of the clonality of any cell lines which survive to outgrowth. This series of VIPS resulted in a total of 570 seeding procedures. Of these, 395 actions were recorded as single cell seeding events, indicated by green circles on the plate map. If single cells survive, and clonal outgrowth succeeds, then any cell lines created from these events are clonal; they have originated from a single parental cell. Other seeding events in this series resulted in two or more cells being seeded in a single well, 167 events, indicated by a red circle with an X on the plate map which were discarded as they were not clonal in origin.

Assessment of rAAV production by SASF clonal cell lines in 125 mL shake flasks

Verified clonal cell lines were moved from 96-well plates to 24-well plates and then to 125 mL shake flasks. Cell line productivity from the surviving clonal cell lines was evaluated by determining rAAV titers at harvest. This step became the bottleneck in assessing and triaging clones for cell line development. Cell lines were transiently transfected with a combination of three plasmids: rAAV eGFP (enhanced green fluorescent protein) transgene, a common reporter used in process development (PD) internal work, an adenoviral helper plasmid and an AAV helper (serotype D) plasmid. Viral titers were determined by quantitative real-time polymerase chain reaction (qPCR). Analyses of rAAV production for a panel of 32 different clonal SASF cell lines is presented in Figure 2. Harvest titers ranged from 1.21 ± 0.12 E+11 DNase resistant particles (DRP)/mL to 2.99 ± 0.03 E+11 DRP/mL. The commercial (control) cell line, which has been used by PD for internal development, produced a harvest titer of 3.2 ± 0.06 E+11 DRP/mL. The wide range in rAAV titers was consistent with the clonality of these cell lines as, clearly, different monoclonal lines will vary in their capacity for manufacturing rAAV. The differences in virus production, illustrated by Figure 2, demonstrated the phenotypic variation that exists among this panel of cell lines. rAAV titer at harvest was a critical functional parameter for selecting a smaller subset of six clones for further intensive characterization. These selected lines are highlighted on Figure 2 (red stars).

rAAV-eGFP serotype D production in SASF clonal lines: scale up and 2L yield assessments

Each of the six candidate clones were batched up to 2000 mL cultures in shake flasks to demonstrate feasibility of flask-scale production and purification. Clones were designated SASF123-5, SASF123-6, SASF124-6, SASF124-7, SASF125-3, and SASF125-4. The optimized manufacturing protocol that had been developed with the PD commercial (control) cell line was used. All cell lines were seeded at 2.50 E+05 cells/mL three days prior to transfection. The cultures were transfected using a transfection reagent (FectoVIR-AAV, Polyplus). All flasks had acceptable cell viability (>97%) on transfection day, indicating that the culture conditions were suitable for all clones.

Flasks were harvested five days post transfection and analyzed for media release of rAAV as well as that retained in the cells. The media was cleared of cells by centrifugation. Analyses of cell pellets revealed that 10–37% of the rAAV (serotype D) remained in the cells. The harvested supernatants were clarified over a 5-inch V100 capsule filter into a membrane (Mini Kleenpak EKV, Pall Corporation), using a standard protocol. The clarified harvests were concentrated and diafiltered using 0.02 m2 tangential flow filtration (TFF) cassettes (Pall Corporation). The recoveries from harvest ranged from 76% to >100%. A >80% recovery from harvest is typical for this TFF process with the PD control cell line. The TFF concentrates were further processed by iodixanol (IDX) ultracentrifugation. The IDX pools were polished by ion exchange chromatography, using 1 mL ion exchange columns.


The initial harvest yields of rAAV-eGFP serotype D ranged between 2.05 E+14 DRP–4.68 E+14 DRP (Figure 3 (A)). The best-producing clone was SASF125-3. As can be seen in Figure 3 (A) and (B), the overall recoveries from harvest to final were in the range of 41–53% as opposed to 20–30% seen with the PD commercial cell line. Qualitative assessment of viral attributes of the purified rAAV from these clones, including drug substance purity, capsid Full:Empty (F:E) ratios, and the in-process recoveries at each step, confirmed that the clones did not present any unusual process characteristics in manufacturing that would have excluded them from further characterization and consideration as final candidates.

Several phenotypic characteristics and process-specific criteria were considered when deciding which SASF cell line would be selected for complete characterization and subsequent transfer for creation of a GMP MCB. Clone SASF125-3 was selected as the final candidate for the clonal suspension cell line. The original designation has been shortened to SASF125; both terms refer to the same cell line. As proof of concept, production with SASF125 was scaled up to a STR 50 bioreactor (Pall Allegro). rAAV-eGFP serotype D was purified to the TFF process stage (Table I). A transfection reagent was used (FectoVIR-AAV, Polyplus). The rAAV titer at harvest was 4.02 E+11 vg/mL. Average rAAV-eGFP serotype D titers at harvest with the PD commercial cell line, at the same scale, were approximately 1.15 E+11 vg/mL (n=4).

Optimization of rAAV manufacturing with the SASF125 clonal cell line

The original in-house PD suspension manufacturing process for rAAV productions at all scales (125 mL flask to STR 2000 bioreactor) was developed for different serotypes by utilizing the DoE strategy to optimize titers and quality (9,13–16). A commercial cell line was used at these earlier stages of development during the process of deriving the clonal HEK293 lines. It is evident that rAAV manufacturing capabilities vary between different HEK293 lineages (Figure 2), a reflection of their different evolutionary trajectories. Recognition of this fact required the use of similar DoE strategies as before to optimize multiple factors critical for rAAV manufacturing. Figure 4 shows representative results from a DoE experiment evaluating the effect of viable cell density at transfection, and the plasmid DNA concentration per 1.0 E+06 cells on rAAV (serotype D) titers. A model was developed using multiple linear regression relating these factors to the vector genome titer, and this model was used to identify optimal ranges for each factor.

Iterative DoEs resulted in an optimized set of conditions for maximizing titers and quality in the SASF125 line. Results from two rounds of DoE are shown in Figure 5 and demonstrate the versatility of the cell line in rAAV manufacturing with different serotypes. This figure summarizes much of the development work that has been accomplished since the isolation of SASF125 and scaling it to the STR 50. As benchmarks, Figure 5 also shows titers obtained from optimized manufacturing conditions with the original commercial cell line. It should be noted that manufacturing conditions and, indeed platform components, are different for the two cell lines. They are different lineages of HEK293 that have been generated under different conditions and thus, as demonstrated, have different manufacturing requirements for optimal rAAV production and purification. There is variability in rAAV yield between different serotypes but the SASF125 outperformed the commercial cell line in five out of the six serotypes investigated to date. Serotypes C and D demonstrate approximately seven-fold increases in rAAV titer.

In summary, a clonal suspension cell line was successfully derived that is suitable for rAAV production. The clonal cell line has been tested with several serotypes, both conventional and engineered. A GMP MCB has been generated, tested, and released for clinical manufacturing of rAAV gene therapy biotherapeutic drugs.


The authors would like to thank the Analytical Development group of Andelyn Biosciences for their constant support with rAAV analytics, especially PCR. The authors also thank Lenore Giannunzio who was manager of the PD group when the SASF cell line development program was initiated.


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About the authors

Michael Mcilhatton, PhD, is a Senior Scientist in the Process Development lab at Andelyn Biosciences. He has a B.Sc. in Biochemistry & Microbiology, and a Ph.D. in molecular virology, from Queen’s University Belfast. His focus is on upstream production in AAV manufacturing, with emphasis on cell line development. He has many years of experience with generating and characterizing both mouse and human primary, tumor, and organoid cell lines.

John Ketz is a Senior Scientist in the Process Development lab at Andelyn Biosciences. He has over 6 years of experience on both upstream and downstream optimization of AAV, its scale up from flasks to STR 2000 bioreactors, associated documentation, and tech transfer. He has a B.S. in Biochemistry and a M.S. in Animal Physiology from West Virgina University, WV.

Sarah Bergman, is a Scientist in the Process Development lab at Andelyn Biosciences. Her focus is on optimization of upstream manufacturing and technical writing. She has over 5 years of experience in AAV manufacturing, its scale up and tech transfer. She has a B.A. in Microbiology from Ohio Wesleyan University, a graduate certificate in Applied Statistics and is on track for Masters of Applied Statistics from Penn State University, PA.

Brittany Hurley, is a Scientist in the Process Development lab at Andelyn Biosciences. Her focus is on AAV manufacturing from small flask to bioreactor operations, including downstream processing, and associated documentation. She has over 5 years of experience in AAV manufacturing, its scale up and tech transfer. She has a B.S. in Biology from The Ohio State University, Columbus, OH.

Byron Carper, Scientist in Process Development at Andelyn Biosciences, has over 6 years of experience working in downstream purification of standard and novel AAV serotypes. He has also been involved in the development a robust, scalable method for purification of AAV productions from 1L to 500L. He has a B.S. in Biomedical Engineering from The Ohio State University.

Andrew Moreo oversees process development as well as preclinical plasmid and viral vector production at Andelyn Biosciences. He leads a diverse organization of interdisciplinary scientists specializing in high throughput manufacturing as well as process development, optimization and scale-up of production platforms. He has over two decades of experience in genetic and molecular research with roles at Syngenta and The Ohio State University. He has a B.Sc. in Biology from Purdue University.

Samir Acharya, PhD, Associate Director of Process Development at Andelyn Biosciences, is responsible for Process and Platform Development, its optimization, characterization, and technology transfer. Prior to Andelyn Biosciences, he has over 25 years of research experience in mechanisms of genomic instability and pathways of cell survival and proliferation. He has authored numerous peer-reviewed scientific publications and book chapters. He has a B.Sc in Chemistry from St. Stephen’s College, University of Delhi; M.Sc. in Biotechnology from Jawaharlal Nehru University, and a Ph.D. in Biochemistry from Indian institute of Science, Bangalore, India.

Article details

BioPharm International®

Vol. 37, No. 5

May 2024

Pages: 11-16


When referring to this article, please cite it as Mcilhatton, M; Ketz, J; Bergman, S; Hurley, B; Carper, B; Moreo, A; Acharya, S. Strategy for Derivation and Optimization of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production. BioPharm International 2024 37 (5).