From Cell Line to Purified Drug Substance: How Therapeutic Antibodies Are Manufactured in Mammalian Cells

Why Mammalian Cells?

Before going into the process itself, one question must be answered: why go to all this trouble with mammalian cells? Would bacteria such as E. coli not be simpler and cheaper?

The answer lies in the "finishing" capacity of the cell. Many therapeutically valuable proteins (antibodies, hormones and enzymes) must undergo complex folding and post-translational modification — glycosylation in particular — before they acquire the correct structure and biological function. Mammalian cells possess a protein-processing system similar to that of human cells and can therefore produce products that closely resemble native human proteins and are both safe and effective. Bacterial systems are unable to carry out these complex modifications, and the proteins they produce may lack activity or provoke an immune response.

For scFv and Fab fragments, however, E. coli expression is a viable option. scFv and Fab contain only the variable regions of the antibody, which are responsible for antigen binding, and lack the Fc region. They therefore do not require glycosylation and are structurally relatively simple, so a prokaryotic system such as E. coli is sufficient for their production.

图1 大哺乳动物细胞工作流程.png

Figure 1. Overview of the mammalian cell-based manufacturing workflow.

The Manufacturing Workflow

1. Upstream Processing — Cell Culture and Antibody Production

The goal of upstream processing is to establish a "cell line" that produces the protein of interest in a stable and efficient manner.

Step 1: Vector construction and cell line development

Gene cloning: Obtain the light-chain (LC) and heavy-chain (HC) gene sequences of the target antibody from hybridoma cells or by gene sequencing, and clone the two genes into a dedicated expression vector.

Cell transfection: Introduce the constructed vector into a selected mammalian host cell. The most commonly used cell lines are:

Human embryonic kidney (HEK293) cells: rapid protein expression and high transfection efficiency, and therefore widely used for transient expression.

Chinese hamster ovary (CHO) cells: the industry gold standard. They perform correct human-compatible post-translational modifications (such as glycosylation) and can be adapted to suspension culture in serum-free medium.

Selection and cloning: Add selection agents to the culture medium (for example the antibiotics G418 or blasticidin). Only cells that were successfully transfected and have integrated the vector — which carries the resistance gene — are able to survive.

From the surviving cell pool, single cells are distributed into 96-well plates by limiting dilution, ensuring that the cells in each well originate from a single progenitor cell and thus forming one clone.

Cell line construction: Screen a number of clones and select those that grow vigorously, give a high antibody yield and show consistent product quality. Expand the selected clones to establish a master cell bank (MCB) and a working cell bank (WCB), which provide a long-term, stable supply of production cells.

Step 2: Cell culture and expansion

This is a stepwise scale-up process designed to generate a sufficient number of cells to produce large quantities of antibody.

Step 3: Antibody recovery

Once cell viability and antibody titre have reached their peak, the cells are separated from the antibody-containing culture supernatant. The supernatant is retained for downstream purification of the antibody.

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. Comparative timelines for the generation of transient and stable protein-producing cell lines.

2. Downstream Processing — Antibody Purification and Polishing

Step 4: Chromatography

This is the most critical step. Capture is achieved by exploiting the specific binding of the antibody Fc region to Protein A, a protein derived from Staphylococcus aureus.

After affinity capture, antibody purity is already high, but a small amount of impurities remains, such as host cell proteins (HCPs) and DNA. Two different chromatographic methods are therefore usually used in series. Examples are ion-exchange chromatography, which separates on the basis of charge (the antibody is positively charged under the chosen conditions while the impurities are negatively charged), and size-exclusion chromatography, which separates according to molecular size.

图3 亲和色谱法.png

Figure 3. Affinity chromatography. (Figure 3. Affinity chromatography)

Step 5: Viral removal

The purified antibody solution is passed through a membrane with a very small pore size (typically 15–20 nm). This pore size is large enough to allow antibody molecules to pass through, yet it physically retains and removes any virus particles that may be present.

In this way, large quantities of active antibody are obtained.

Service Capabilities

Tek Biotech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform based on phage display and yeast display technologies. We provide scientists worldwide with high-quality monoclonal antibody development services covering a wide range of formats, including scFv, VHH and Fab, and are able to develop antibodies with a variety of functional and structural characteristics (including but not limited to neutralizing antibodies, conformation-specific antibodies and cross-reactive antibodies). Tek Bioscience also offers supporting services such as downstream antibody expression and validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, meeting the antibody drug development needs of customers across a broad range of applications.

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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