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Clone selection is one of several key steps in a successful stable cell line development (CLD) process. To overcome the shortcomings of traditional manual cloning and screening processes, we have successfully integrated two high-throughput cloning methods: FACS and ClonePixTM into our CLD workflow. Both methods are highly robust, and utilize a primary clone screening step based on high expression of the protein of interest.

A side by side comparison of ClonePixTM with traditional limiting dilution cloning (LDC) was performed using a same monoclonal antibody expressing cell pool. ClonePixTM is an automated selective clone selection method compared to highly labor-intensive and random LDC method. In addition, we present case studies using FACS cloning in combination with CloneSelectTM Imager which outline an approach for identifying high-producing clones with only one round of cloning while ensuring the monoclonality, which significantly reduces the CLD timeline. Overall, the studies presented here aim at demonstrating high-throughput cloning technologies that reduce cost, labor and timeline for CLD and improve clone selectivity to ensure the identification of high producing clones.

Abstract

Kitty Agarwal, Rich Harper, Brian Davies, Amy Venturini, Abhinav Shukla and Ying Huang

Cell Line Development, KBI Biopharma Inc., Durham, North Carolina 27704.

Integrating High-Throughput Cloning into Cell Line Development

Traditional vs. High-throughput Cloning

www.kbibiopharma.com

We used the same mAb producing CHO K1 cell pool for cloning and clone screening for both LDC and ClonePixTM. For LDC, cells were seeded at 0.5cells/well in 96-well plates and wells with >10% confluence were scaled up to 24-well plates in ~ 3 weeks. Monoclonality and growth were monitored using CloneSelectTM Imager (CSI). For ClonePixTM, cells were seeded in semisolid media containing CloneDetect (FITC labeled antibody). 10 days post-seeding high producing clones were picked using CLonePixTM based on shape, size, proximity and exterior mean fluorescence intensity into 96-well plates. Following titer assessment, high producing clones were scaled up to 24-well plate. For both the cloning techniques, clones were further scaled up to 6-well plates and shake flasks based on productivity. Titers were measured using ELISA assay.

For FACS we present multiple case studies of single cell cloning and clone screening of mAb producing CHO S cells to select high-producing monoclonal clones (mAb X and Y) using FACSJazzTM cell sorter. For each mAb pool, single cells were sorted based on high fluorescence signal into 96-well plate. Following titer assessment high producing clones were scaled up to 24-well plate. Monoclonality was verified using CSI. The same work flow was followed as LDC and ClonePixTM from here on. Top 3 clones mAb X clones were assessed for productivity with different feeds and varying feed percentages. Titers for FACS studies were measured using high-throughput methods: OctetTM and Pro A HPLC.

Conclusions Cell line selection were run in parallel using conventional LDC and high-throughput automated ClonePixTM. The productivity of the selected cell lines from the two methods was comparable. ClonePixTM successfully generated high yielding clones.

ClonePixTM and FACS offer the advantage of clone screening based on productivity early on during clone selection, thus increasing the probability of generating high-producing clones by screening more number of clones in a shorter duration compared to LDC.

FACS can successfully sort and clone cells with favorable attributes and clonality can be verified using the CloneSelectTM Imager.

Further improvement of strategies to reduce time lines and increase productivity are ongoing, which include direct selection, amplification and cloning with ClonePixTM and pool enrichment using FACS, as well as incorporation of other high-throughput methods like ambrTM for clone screening.

Single Cell Cloning using FACS

Figure 2: Cell growth (i) and monoclonality (ii) of clones recovered from LDC

were monitored using CSI. Starting from 2 hours post-seeding, CSI was used to

capture cell images on Days 0, 1, 2, 7, 14 and 19. Well Images (ii) of

representative clone presented here showing the clone growth.

Figure 1: Cloning using ClonePixTM and FACS involves selecting cells based on high expression of mAb opposed to Limiting Dilution Cloning (LDC) in which cells are seeded into 96-well plates to ensure single cells per well.

LDC vs. ClonePixTM: Suspension Culture Assessment

Figure 4: Top 30 clones were further assessed for productivity in a 7 day batch

culture. (i) On average, clones identified by LDC and ClonePixTM had 1.8-fold and

2.5-fold higher mAb productivity compared to the parent pool respectively. (ii)

Clone ranking across different screening stages. For ClonePixTM, 9 of the top 10

clones in shake flask were among the top 30 clones at the 6-well plate stage,

indicating a good correlation between clone rankings across the screening stages.

Top 5 ClonePixTM clones were further evaluated for mAb productivity using different

feeding strategies.

Shake Flask Batch Culture

Fed Batch Productivity Assessment

Figure 5: Top 5 clones by

ClonePixTM were assessed using

two different feeding strategies.

mAb productivity for both the

schemes was found to be similar.

Figure 6: Top mAb pools (mAb X and Y; representative mAb X FACS profile

shown in (i)) selected based on titer and FACS profile for single cell cloning. Cell

growth and monoclonality were monitored using CSI. CSI cell images were

captured on Days 0, 1, 2, 3, 7, and 14. Outgrown clones were assessed for titer.

High yielding clones were scaled up to 24-well plate followed by expansion of top

~ 60 clones to 6-well plate and further top 30 clones to shake flask based on

absolute and growth-normalized titers.

Figure 8: Top 10 clones were assessed for productivity with simple fed batch.

Viable Cell Density (data not shown) and harvest day titer were measured. Highest

producing clone titers were 0.44g/L (mAb X) and 0.33g/L (mAb Y).

Figure 9: Top 3 mAb X clones were assessed for productivity with different feeding

strategies. One of the feeding strategies (Scheme 2) provided the highest titer of

0.94g/L for clone2. Scale up of the clones to bioreactors (3L) gave high titer (1.0-

1.3g/L) for clone2 indicating the scalability of clones generated by FACS.

Limiting Dilution Cloning

ClonePixTM: Screen for High Yielding Clones

Figure 3: ClonePixTM images on Day10 post-seeding (i) in white and fluorescent

light indicate presence of high yielding clones. Post-picking cell growth was

monitored on Days 2 and 6 using CSI (ii and iii). ClonePixTM allows clones to be

selected based on high mAb productivity early on in the clone screening process .

Titer of mAb X Clones Titer of mAb Y Clones

Figure 7: mAb X and mAb Y clones were assessed for productivity at each

screening stage in static culture using high-throughput OctetTM system, to select for

high producing clones. Top 30 clones were scaled up to shake flask.

Fed Batch Assessment of Top 3 mAb X Clones

Simple Fed Batch Culture of Top 10 mAb X and mAb Y Clones

LDC vs. ClonePixTM: Static Culture Assessment

(i)

(ii)

Static Culture screening of mAb X and mAb Y Clones from FACS

mAb X and mAb Y Clone Selection in Shake Flask