Expression of Recombinant Antibodies in Mammalian Cells: Core Technologies – Comprehensive Analysis of Host Selection and Purification Strategies

1. Expression Host:

The primary rationale for selecting mammalian cells lies in their capability to execute a series of complex biological processes essential for the proper folding, assembly, and post-translational modification of antibodies. Among these, glycosylation is pivotal, particularly N-linked glycosylation on the Fc region, which directly influences the antibody's half-life, immunogenicity, and effector functions (such as antibody-dependent cell-mediated cytotoxicity [ADCC] and complement-dependent cytotoxicity [CDC]).

Currently, the mainstream mammalian host species widely adopted in the industry and extensively validated clinically include the following:

China hamster ovary cells (CHO): The most widely used and mature mammalian cell host in the fields of recombinant protein and therapeutic antibody production, without exception. This is attributed to their well-established industrial platform, excellent growth in chemically defined media—facilitating process control and product quality consistency—good tolerance to scale-up cultivation, and, most importantly, their inability to synthesize terminally linked sialic acid α-2,6-galactose and N-hydroxyacetylneuraminic acid (which are immunogenic in humans). However, the construction of these cell lines is time-consuming, and the expressed products exhibit microscopic heterogeneity.

Human embryonic kidney 293 cells (HEK293): These cells can rapidly produce antibody proteins ranging from milligrams to grams within days to a week; their transient expression levels are typically higher than those of CHO cells; and they theoretically provide glycosylation modifications closest to those of natural human antibodies. However, the maturity of this expression platform is inferior to that of CHO cells, and the cost of scale-up is relatively high.

Other: Mouse myeloma cells, such as NS0 and Sp2/0. These served as early industrial standards; however, their use has declined with the increasing sophistication of CHO and HEK293 cell platforms.

2. From the crude extract to a high-purity product

The objective of downstream purification is to isolate the target compound with high purity, high yield, and high activity. A mature industrial-scale purification process typically follows the sequence of "capture → refinement → polishing."

Step 1: Capture – Protein A affinity chromatography

Principle: Protein A, derived from Staphylococcus aureus, binds to the Fc segment of antibodies with high affinity and specificity. By immobilizing Protein A onto a chromatographic medium, an affinity chromatography column is formed. Upon loading the sample, the antibody is specifically "captured" on the column, enabling a one-step process to increase antibody purity from approximately 1–5% to over 95%. However, this method is relatively costly and may require additional testing for antibodies that have undergone Fc engineering modifications or belong to certain rare subtypes.

Step 2: Ion exchange chromatography and hydrophobic interaction chromatography

Antibody aggregates with immunogenicity risks, host cell proteins that may trigger immune responses, host cell DNA with potential carcinogenic risks, shed Protein A, and viruses may still remain after undergoing Protein A affinity chromatography.

The following methods are typically used for further removal.

The ion-exchange permeation mode is the most commonly employed strategy. The pH and conductivity of the sample are adjusted to impart a net positive or weakly negative charge, as most HCPs, DNA, and viruses carry strong negative charges. When the sample flows through the anion exchange column, antibodies do not bind and pass directly through, whereas strongly negatively charged impurities are captured by the column, achieving efficient removal.

Cation exchange binding-elution mode: Can be used to separate antibody charge heteromers (e.g., those resulting from modifications such as deamidation or oxidation) and further remove aggregates and HCPs.

Hydrophobic interaction chromatography: This is the preferred method for removing antibody aggregates. Due to their larger hydrophobic surface area, aggregates exhibit stronger binding affinity than monomers, thereby enabling effective separation.

The most critical step is virus removal, which is a regulatory requirement. This can be achieved by incubating the sample under pH conditions (3.5–3.9) for a specified duration to effectively inactivate enveloped viruses; alternatively, viral particles with a physical size of 15–20 nm can be removed using size exclusion filtration.

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Tek Biotechnology (Tianjin) Co., Ltd. has established a comprehensive recombinant antibody expression platform using mammalian cells, dedicated to providing high-quality recombinant antibody expression services for scientists worldwide. This platform leverages the advantages of the mammalian cell expression system to ensure the delivery of high-quality recombinant antibodies with superior biological activity. We offer robust support for clients' research projects and the development of novel antibody-based therapeutics.

References ：

[1] Eon-Duval A, Broly H, Gleixner R. Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach. Biotechnol Prog. 2012 May-Jun;28(3):608-22.

[2] Rathore AS. Follow-on protein products: scientific issues, developments and challenges. Trends Biotechnol. 2009 Dec;27(12):698-705.

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