利用瞬时 CHO 表达系统在培养中捕获抗体

Jing Zhou; Lauren Gebhardt; Molica Abel; Audrey Vogt; Bo Hee Shin; Ana Santos; Jerome Puginier; Florian M. Wurm; Maria J. Wurm; Sarah L. Herrick-Wagman; Adedolapo Adeniyi; Guoying Grace Yan; Sean Lim; Laura Lin; Aaron M. D’Antona; Xiaotian Zhong

doi:10.21769/BioProtoc.5694

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Peer-reviewed

In-Culture Antibody Capture Using Transient CHO Expression Systems

利用瞬时 CHO 表达系统在培养中捕获抗体

JZ Jing Zhou ^*

LG Lauren Gebhardt ^*

SH Sarah L. Herrick-Wagman

AD Aaron M. D’Antona email

XZ Xiaotian Zhong ^§ email

(*contributed equally to this work, § Technical contact) 发布: 2026年06月05日第16卷第11期 DOI: 10.21769/BioProtoc.5694 浏览次数: 298

评审: Neha NandwaniNidhi MenonAnonymous reviewer(s)

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Cover of Antibodies, featuring study using the protocol.

Dec 2025

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Abstract

Antibody therapeutics have demonstrated transformative impacts on improving the quality of life of millions of patients, whereas advances in antibody discovery technologies have imposed a significant production challenge for the generation of a large diversity of therapeutic antibody candidates. A demand for the rapid production of dozens of purified antibodies in 10-mg quantities is entailed for functional screening and molecular assessment studies. Here, we present a robust semi-automated production protocol that bridges the gap between miniaturized high-throughput screenings and conventional custom-scale workflows. This methodology and workflow utilize a simple high-titer transient Chinese hamster ovary (CHO) cell host–CHO4Tx^® expression system, a procedure of magnetic protein-A bead in-culture antibody capturing, and a semi-automated purification process with the GenScript AmMag^TM SA Plus system. This production protocol has been proven to be robust and valuable for the routine production of dozens of antibody constructs per week in sufficient quality and quantity for cell-based and biophysical studies.

Key features

• This protocol utilizes the high-titer CHO4Tx^® cell line, which helps increase protein production throughput by decreasing culture volume and eliminating DNA/transfection reagent complexation.

• This protocol simplifies and expedites protein production processes by eliminating time-consuming and costly steps such as cell centrifugation, medium filtration, and medium column loading.

• Although we present this workflow using CHO4Tx^®, magnetic bead purification methods can be applied to other CHO cell lines and human embryonic kidney-293 cells.

Keywords: Transient gene expression (瞬时基因表达)

CHO4Tx^® (CHO4Tx^®)

In-culture antibody capturing (培养中抗体捕获)

AmMag^TM Protein-A magnetic beads (AmMag^TM Protein-A magnetic beads)

Semi-automation purification (半自动化纯化)

GenScript AmMag^TM SA-Plus (GenScript AmMag^TM SA-Plus)

Graphical overview

Background

More than 200 antibody therapeutics have received worldwide approval since the invention of hybridoma technology by Kohler and Milstein in 1975 [1], providing tremendous therapeutic benefits to patients. The modern era of antibody therapeutics, launched by high-quality display technologies, immunization, and single-cell isolation [2–5] and by de novo protein synthesis [6–10], has generated many potential antigen binders. Efficient hit evaluations have created an urgent need for rapidly generating thousands of isolated and engineered antibody proteins with an unknown expression performance via high-throughput screening methods, and a demand for a mid-scale production process for a sizable number of follow-on molecules. Rapid and high-fidelity antibody production is also increasingly important for enabling early mechanistic and safety evaluation in discovery pipelines, which integrates advanced culture models, functional signaling readouts, imaging, and biomarker-based assays [11,12]. Transient gene expression (TGE) technologies in human embryonic kidney (HEK)-293 and Chinese hamster ovary (CHO) cells [13–17], exploiting episomal plasmid DNAs for protein synthesis without chromosomal integrations, can routinely provide proteins in days for screening assays, functional investigations, and preclinical analysis during discovery and early development. HEK293 cells exhibit high transfectability and reproducibility, whereas CHO cells possess a unique advantage of uncovering potential issues for candidate molecules at the early stages of drug development [18–25].

Antibody purification, driven by the increasing speed in antibody discovery, proceeds with automation and high throughput [26–29]. While miniaturized high-throughput (HTP) plate-based or tube-based purification has been widely adopted for material generation to meet the small protein requirements for relatively simple screening, this HTP method cannot provide enough antibodies with suitable purity for more sophisticated assays. Substantially larger quantities of purified antibodies of at least 10 mg are typically required for biophysical characterization and in vitro functional testing. In addition, typical antibody production procedures involve laborious and time-consuming steps such as clarification of cells from cell culture via centrifugation and/or filtration, as well as proA column loading of conditioned media for protein capturing. The cell separation step can potentially increase host cell protein (HCP) levels and proteolytic activity in the clarified materials.

Our protocol offers a semi-automated novel production methodology and workflow by taking advantage of a high-titer transient CHO expression system for 100 mL scale productions to meet the demand for rapid production on the 10 mg scale. A magnetic proA bead in-culture antibody-capturing process for transiently transfected CHO cells, coupled with the utilization of the GenScript AmMag^TM SA Plus semi-automation system, can eliminate the steps of cell clarification, filtration, and medium loading in a throughput manner. This production protocol could support the weekly generation of dozens of antibody proteins with adequate quality and quantity for drug discovery characterizations [30].

Materials and reagents

Biological materials

1. CHO4Tx^® (Magellan Biologics, origin: CHOExpress^®, https://magellanbiologics.com/products/, info@magellanbiologics.com)

Reagents

1. CHO4Tx^® cultivation medium (CHO4Tx^® CM) (store at 4 °C, 6-month shelf-life) (Magellan Biologics, https://magellanbiologics.com/products/, info@magellanbiologics.com)

2. CHO4Tx^® transfection medium (CHO4Tx^® TM) (store at 4 °C, 6-month shelf-life) (Magellan Biologics, https://magellanbiologics.com/products/, info@magellanbiologics.com)

3. CHO4Tx^® production medium (CHO4Tx^® PM) (store at 4 °C, 6-month shelf-life) (Magellan Biologics, https://magellanbiologics.com/products/, info@magellanbiologics.com)

4. AmMag^TM Protein A magnetic beads (Genscript, catalog number: L00695-80)

5. Glycine hydrochloric acid (HCl) (Sigma, catalog number: G2879)

6. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S640)

7. 10 M sodium hydroxide (NaOH) (Fisher Scientific, catalog number: SS255)

8. Calcium–magnesium-free phosphate buffered saline (PBS-CMF) (Thermo Fisher, Gibco, catalog number: 21600-044)

9. Tween 80 (Sigma, catalog number: P1754)

10. 5 M hydrochloric acid (HCl) (Baker, catalog number: 5618-02)

11. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher, Gibco, catalog number: 14190144)

12. L-Glutamine (200 mM) (Thermo Fisher, Gibco, catalog number: 25030081)

13. 1 M HEPES pH 8.0 (Teknova, catalog number: H1090)

14. Reverse osmosis deionization (RODI) water

Solutions

1. Elution buffer pH 3.0 (see Recipes)

2. Wash buffer (see Recipes)

3. CHO4Tx^® cultivation medium (CHO4Tx^® CM) (see Recipes)

Recipes

1. Elution buffer pH 3.0

Reagent	Final concentration	Quantity or volume
Glycine HCl	150 mM	33.46 g
NaCl	40 mM	4.68 g
10 M NaOH (titrant)	120 mM	24.00 mL
RODI water	n/a	1.976 L
Total (optional)	n/a	2 L

2. Wash buffer

Reagent	Final concentration	Quantity or volume
PBS-CMF	99.98%	9.55 g
Tween 80	0.02%	0.213 g
5 M HCl (titrant)	7.9 mM	1.58 mL
RODI water	n/a	1 L
Total (optional)		1,001.58 mL

3. CHO4Tx^® CM

Reagent	Final concentration	Quantity or volume
CHO4Tx^® CM	n/a	1 L
200 mM L-Glutamine	4 mM	20 mL

Laboratory supplies

1. Thomson Instrument optimum growth 250 mL flask w/ vent cap, sterile (Thomson Instrument Company, catalog number: 931111-PFZ)

2. Corning^® Erlenmeyer cell culture 500 mL flask (Corning^®, catalog number: CLS431144)

3. Thomson Instrument optimum growth 5 L flask (Thomson Instrument Company, catalog number: 931116)

4. Corning^® conical-bottom centrifuge tubes, graduated, sterile (Corning^®, catalog number: 430291)

5. Corning^® 250 mL PP centrifuge tubes with plug seal cap, sterile (Corning^®, catalog number: 430776)

6. Corning^® 500 mL PP centrifuge tubes with plug seal cap, sterile (Corning^®, catalog number: 431123)

7. Optifit pre-sterilized racked 200 μL pipette tips (Sartorius, catalog number: 790201)

8. Optifit pre-sterilized racked 350 μL pipette tips (Sartorius, catalog number: 790351)

9. Optifit pre-sterilized racked 1,000 μL pipette tips (Sartorius, catalog number: 791001)

10. Optifit pre-sterilized racked 5,000 μL pipette tips (Sartorius, catalog number: 780305)

11. Eppendorf Tube^®, 5 mL (Eppendorf, catalog number: EP0030119487)

12. Snap-cap low-retention microcentrifuge tubes, 1.5 mL (ThermoFisher Scientific, catalog number: 3451PK)

13. Nunc^TM 5 mL serological pipette (ThermoFisher Scientific, catalog number: 170355N)

14. Nunc^TM 10 mL serological pipette (ThermoFisher Scientific, catalog number: 170356N)

15. Nunc^TM 25 mL serological pipette (ThermoFisher Scientific, catalog number: 170357N)

16. Nunc^TM 50 mL serological pipette (ThermoFisher Scientific, catalog number: 170376N)

17. EZFlow sterile PEB syringe filters, 0.22 μm sterilizing membrane, 25 mm (Foxx Life Sciences, catalog number: 50-104-9912)

18. Safety syringe, 3 mL (Sol-Care^TM, catalog number: 22-024-411)

19. Biosensor/Protein A (ProA) (Sartorius, catalog number: NC9490476)

20. 96-well, cell culture-treated, flat-bottom, half-area microplate (Greiner Bio, catalog number: 07-000-095)

21. 96-well deep-well plates, square v-bottom (BrandTech Scientific, catalog number: 14-380-947)

22. Reagent reservoir, 10 mL (Fisherbrand^TM, catalog number: 01-670-461)

23. Disposable roller bottles (Corning, catalog number: 09-761-113)

24. Corning^® Erlenmeyer cell culture 125 mL flask (Corning, catalog number: CLS431143)

25. HiPrep^TM desalting columns with Sephadex^TM G-25 resin (Cytiva Life Sciences, catalog number: 17508701)

26. Beckman Coulter Vi-CELL sample vials, 4 mL (ThermoFisher Scientific, catalog number: 50-010-41)

27. Corning^® 50 mL centrifuge tubes (Millipore Sigma, catalog number: CLS430290)

Equipment

1. AmMag^TM SA Plus semi-automated purification system (Genscript, catalog number: L01013)

2. AmMag^TM MR magnetic rack (Genscript, catalog number: L00723)

3. Kuhner ISF1-Z incubator shaker (Kuhner Inc, model: ISF1-Z)

4. Incubator tray with sticky strips, F-size tray, 8 sticky strips (Kuhner Inc., catalog number: 105200)

5. Beckman Coulter Allegra X-12R refrigerated centrifuge (Beckman Coulter, catalog number: BE-AX12R)

6. Octet RED96e (Sartorius, model: Octet RED96e)

7. SterilGARD^® e3 Class II biosafety cabinet (The Baker Company, model: SterilGARD e3)

8. Vi-CELL XR Cell Viability Analyzer (Beckman Coulter, model: Vi-CELL XR)

9. Proline^® Plus mechanical pipette, single-channel 20–200 μL (Sartorius, catalog number: 728060)

10. Proline^® Plus mechanical pipette, 8-channel 30–300 μL multichannel pipette (Sartorius, catalog number: 728140)

11. Proline^® Plus mechanical pipette, single-channel 100–1,000 μL (Sartorius, catalog number: 728070)

12. Proline^® Plus mechanical pipette, single-channel 5,000 μL (Sartorius, catalog number: 728580)

13. S1 pipette fillers (ThermoFisher Scientific, catalog number: 9541)

14. NanoDrop^TM One/One^C microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, catalog number: ND-ONEC-W)

15. Tecan Fluent-480 liquid handler (Tecan, model: Fluent-480)

16. AKTA Pure^TM chromatography system (Cytiva Life Sciences, model: AKTA Pure^TM 150)

Software and datasets

1. FortèBio Data Acquisition (Sartorius, Version 7.1)

2. FortèBio Data Analysis (Sartorius, Version 7.1)

3. UNICORN^TM Control Software (Cytiva Life Sciences, Version 7.10)

Procedure

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稿件历史记录

提交日期: Jan 7, 2026

接收日期: Mar 24, 2026

在线发布日期: Jun 1, 2026

出版日期: Jun 5, 2026

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Zhou, J., Gebhardt, L., Abel, M., Vogt, A., Shin, B. H., Santos, A., Puginier, J., Wurm, F. M., Wurm, M. J., Herrick-Wagman, S. L., Adeniyi, A., Yan, G. G., Lim, S., Lin, L., D’Antona, A. M. and Zhong, X. (2026). In-Culture Antibody Capture Using Transient CHO Expression Systems. Bio-protocol 16(11): e5694. DOI: 10.21769/BioProtoc.5694.