How Are Antibody Drugs Produced? A Deep Dive into Mammalian Cell Culture and Purification Processes

From PD-1 inhibitors for cancer treatment to TNF-αantagonists for autoimmune diseases, these cutting-edge drugs have become widely recognized. However, how are these structurally complex antibody drugs actually produced? The answer lies in a shared manufacturing facility—mammalian cells.

Why are mammalian cells used?

Before delving into the process, it is essential to address this question: Why go to such great lengths to use mammalian cells? Is using bacteria (such as Escherichia coli) not simpler and more cost-effective?

The answer lies in the "fine-processing" capability of proteins. Many therapeutically valuable proteins (such as antibodies, hormones, and enzymes) require complex folding and modifications (particularly glycosylation) to achieve their correct structure and biological function. Mammalian cells possess a protein processing system similar to that of human cells, enabling the production of safe and effective products highly analogous to natural human proteins. In contrast, bacterial systems are incapable of performing these complex modifications, resulting in proteins that may lack activity or trigger immune responses. However, ScFv or Fab fragments can also be expressed using Escherichia coli: these fragments contain only the variable regions of antibodies responsible for antigen binding, with the Fc segment removed. Consequently, they do not require glycosylation and have a relatively simple structure, making prokaryotic systems such as E. coli well-suited for their production.

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detailed procedures

1. Upstream process – Cell culture and antibody production

Our goal is to establish a "cell line" capable of stably and efficiently producing this protein.

Step 1: Vector Construction and Cell Line Development

gene clone ：

Obtain the light chain and heavy chain gene sequences of the target antibody from hybridoma cells or via gene sequencing. Clone these two genes into specific expression vectors.

Cell Transfection:

The constructed vector is introduced into the selected mammalian host cells. The most commonly used cell lines are:

Chinese hamster ovary (CHO) cells: The gold standard in industry, as they can perform correct humanized protein post-translational modifications (such as glycosylation) and are suitable for suspension serum-free culture.

Human embryonic kidney cells: These cells exhibit rapid protein expression and high transfection efficiency, making them commonly used for transient expression.

Filter and Clone:

By adding screening agents (such as antibiotics like G418 or rice blast fungicide) to the culture medium, only cells that have successfully undergone transfection and integrated the vector (containing the resistance gene) will survive.

From the surviving cell population, individual cells were distributed into 96-well plates using the limited dilution method to ensure that each well contained cells derived from a single progenitor cell, thereby forming a clone.

Cell Line Construction:

From numerous clones, several that exhibited vigorous growth, high antibody yield, and stable quality were selected. These optimized clones were amplified to establish a primary cell bank and a working cell bank for long-term, stable supply of production cells.

Step 2: Cell Culture and Expansion

This is a stepwise amplification process aimed at obtaining a sufficient number of cells to produce large quantities of antibodies.

Step 3: Obtaining antibodies

When cell viability and antibody yield reach their peak, the cells are separated from the antibody-containing culture supernatant, with the supernatant retained for subsequent antibody purification.

图2 瞬时和稳定蛋白质生产细胞系生成的比较时间表.png

Figure 2: Comparison of timelines for instantaneous versus steady-state protein production by cell lines

2. Downstream Process – Antibody Purification and Refinement

Step 4 – Chromatographic Method

This is the most critical step, involving crude purification by leveraging the specific binding capability of the antibody Fc region to Protein A (a protein derived from Staphylococcus aureus).

After affinity capture, the antibody exhibits high purity but still contains trace impurities such as host cell proteins and DNA. Typically, two distinct chromatographic methods are employed in tandem, including ion exchange chromatography (which separates antibodies based on their positive charge while impurities carry negative charges) and molecular exclusion chromatography (which separates molecules according to their size).

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Figure 3 Affinity Chromatography

Step 5 – Virus Removal

The purified antibody solution is passed through a filter membrane with an extremely small pore size (typically 15–20 nm). This pore size is sufficient to allow antibody molecules to pass through, but is capable of physically retaining and removing any potentially present viral particles.

This method yields a substantial quantity of active antibodies.

Tek Bio (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform leveraging phage display and yeast display technologies. This platform provides high-quality development services for various types of therapeutic monoclonal antibodies—including scFv, VHH, and Fab—for scientists worldwide, as well as the capability to develop diverse functional structural antibody variants (including but not limited to neutralizing antibodies, conformation-specific antibodies, and cross-reactive antibodies). Additionally, Tek Bio offers complementary downstream services such as antibody expression and validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, thereby meeting the diverse needs of clients across all sectors of antibody-based drug development.

References

[1] Dumont J,Euwart D,Mei B, et al. Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol. 2016;36 (6):1110-1122.

[2] Gupta K,Parasnis M,Jain R, et al. Vector-related stratagems for enhanced monoclonal antibody production in mammalian cells. Biotechnol Adv. 2019;37 (8):107415.

[3] Ecker JW,Kirchenbaum GA,Pierce SR, et al. High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines. Vaccines (Basel). 2020;8 (3):null.

[4] Broer LN,Knapen DG,de Groot DA, et al. Monoclonal antibody biosimilars for cancer treatment. iScience. 2024;27 (6):110115.

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