To achieve over 5 fold of human IgG protein detection signal enhancement with 1 round of CloneDetect K8495 assay optimization

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Paula Zadek | Senior Field Application Scientist III | Molecular Devices Carola Mancini | European Field Application Scientist | Molecular Devices Shan Liu | Product Manager for Reagents and Consumables | Molecular Devices

Introduction

Protein G (Pro G) is a large cell surface binding protein that has a repeating 55-residue domain that binds with high affinity to the FC region of IgG¹. Together with Protein A (Pro A), binding to large amounts of IgG at near physiological pH and ionic strength, these two proteins are extensively used for the purification of IgG antibodies² in the Biopharma manufacturing process. Pro G binding can stabilize the complex structure formed between recombinant CloneDetect K8495 and the secreted IgG antibody proteins thereby creating a complex with enhanced detection sensitivity. Here we exploit that property of Pro G versus that of Pro A to increase the fluorescence detection on the ClonePix® System. The ClonePix images and picks mammalian cell colonies based on their productivity of secreted antibodies or cell surface proteins with a variety of measures that include the secreted or surface binding proteins fluorescent signal³ . Utilizing a combination of imaging and robotics, ClonePix System screens for clonality and productivity simultaneously prior to collecting the highest producing clones in a rapid one-step process (Figure 1).

The purpose of this study was to explore the strategies to increase the detection of the fluorescent signal for optimized colony selection of the best producing cell lines in a CHO cell model, this may be applied to other cell line models such as HEK293 or hybridoma cell lines SP2/0, NS0/1 that are commonly used for therapeutic monoclonal IgG antibodies production.

Figure 1. ClonePix Systems image, analyze, rank and pick selected colonies in 1-step process to increase cell line development workflow throughput and process efficiency

Materials and methods

Prepare stock solutions for recombinant Pro G and Pro A

Add 10 ml of sterile cell culture grade water or DPBS solution to each vial to prepare 0.5 mg/ml stock solution, mix and wait for 10–15 mins in room temperature for complete hydration before filter the stock solution through 0.2 µm PES filter and make aliquots. Store aliquots at 4°C for up to 4 weeks or long term storage at -80°C.

Table 1. Materials used in this study

Cell culture, plating and imaging

CHO cell line model with low human IgG expression (MD) was maintained in XP Media CHO Growth A with 8 mM L-Glutamine and passaged twice a week. 100 µL of log stage cell suspensions at a density of 25,000 – 50,000 cells/ml (final plating density is \~250 – 500 cells per ml) were added to 9.9 mL premixed semi-solid media with the following components:

• 9.0 ml CloneMedia CHO Growth A K8840
• 0.4 ml L-Glutamine
• 0.2 ml CloneXL CHO ACF supplement K8525
• 0.2 ml XP Media CHO Growth A without L-Gln K8860
• 0.1 ml Recombinant CloneDetect K8495

Add different concentration ( 0.0 µg/ml, 0.5 µg/ml, 1.0 µg/ml) of recombinant Pro A or Pro G to the mixed media, using the stock solution (0.5 mg/ml) prepared before.

The final mixed semi-solid media containing supplements, cells, CloneDetect K8495, and either Pro A or Pro G was plated at 2.0 ml/well into each well of Greiner Bio-One CellStar 6-well plate. Space between wells was filled with sterile water to minimize evaporation. Plates were placed in an incubator with > 85% humidity, 5% CO₂ at 37°C. The incubation time and optimal imaging timepoint will be determined experimentally. In this study, the imaging and analysis on the ClonePix system were performed on Day 13 post plating (Figure 2).

Results and discussion

Recombinant monoclonal CloneDetect K8495 (animal origin-free) is preferred for applications required to satisfy highest regulatory demands. When added to semi-solid media at the moment of plating, CloneDetect K8495 diffuses freely, creating immunoprecipitation complexes with target immunoglobulins as they are secreted from the colonies. These complexes generate localized fluorescent signals that, under optimized assay conditions, are proportional to the amount of IgG secreted by a specific clone at the real-time of the IgG quantification conducted. However, due to its monoclonality and high specificities with single binding site within the human IgG Fc region, if the clones to be screened have low levels of expression of the target IgG molecules, assay optimization is required to achieve the optimal results.

In this study, we have explored the usage of recombinant Pro A or Pro G combined with CloneDetect K8495 to enhance the Fluorescent signal for IgG detection/ quantification. The results have shown that adding Pro G at the final concentration of 1.0 µg/ml produced the best outcome with over 5 fold fluorescent signal enhancement, comparing to the control assay without adding Pro G optimizer (Table 2, Figure 3).

How to screen and select clones of interest with high specificity and high efficiency is known as one of the bottlenecks for therapeutic antibody engineering and cell line development⁴. There are many factors that will impact the detection sensitivity and clone screening process: 1) low levels of target protein expression 2) misfolded target protein trapped inside the cells 3) low binding affinity of target protein to detection reagent 4) unstable bindings of target protein to detection reagents.

The combination of Pro G and CloneDetect K8495 amplified the fluorescent signal and improved detection, due to the stabilization of the complexes formed by the target protein, Pro G and CloneDetect reagent. This approach can be applied to a variety of cell line models that express proteins that bind to the stabilized complex formed with CloneDetect, and can be imaged, analyzed and picked by the ClonePix system.

Figure 2. One round of K8495 assay optimization increased the fluorescent signal by over five folds as measured using Sum Total Intensity. Sum Total Intensity is defined as the Sum Fluorescent Intensity of All Pixels of the colony divided by # of Pixels within the colony boundary + halo boundaries with halo determined as 3 x colony diameter ( see Appendix A for ClonePix parameters)

Table 2. CloneDetect K8495 was diluted 100 times, together with optimizer Pro G or Pro A at various final concentration (0, 0.5, 1.0 µg/ml), were added to the semi-solid media with cells on plating date. Each data point represents the mean value of florescence intensity from 3 replicates (n=3), ≥120 colonies measured by ClonePix system on day13 post plating

Figure 3. Each date point represents the mean value of Sum Total Intensity (FITC500ms) from 3 replicates. All assays were conducted on the same cell population. Adding Pro G 1.0 µg/ml to K8495 assay produced the optimal outcome at 5.4-fold increase in the fluorescent signal compared to the addition of Pro A at a 2.3-fold signal increase

Appendix A

ClonePix Parameter setting reference

References

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2. Jordan B. Fishman and Eric A. Berg. Protein A and Protein G Purification of Antibodies. Cold Spring Harb Protoc 2019; doi:10.1101/pdb. prot099143.
3. Dharshanan S, Hung CS. Screening and subcloning of high producer transfections using semisolid media and automated colony picker. Methods Mol Biol. 2014;1131:105-12. doi: 10.1007/978-1-62703-992-5_7. PMID: 24515462.
4. Doerner A, Rhiel L, Zielonka S, Kolmar H. Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett. 2014 Jan 21;588(2):278-87. doi: 10.1016/j.febslet.2013.11.025. Epub 2013 Nov 26. PMID: 24291259.

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