Nanobody Mammalian Cell Expression-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Nanobody Mammalian Cell Expression

Mammalian cells are the best expression system for producing functional antibodies, and most therapeutic antibodies are expressed using mammalian cells. Currently, all monoclonal antibody drugs approved for human use are produced by CHO, SP2/0, NSO and other cells. On the one hand, monoclonal antibody production requires eukaryotic cell expression to ensure the integrity of its function. On the other hand, antibodies produced by mammalian cells have low immunogenicity and high safety. CHO cells have a short growth cycle and can adapt to suspension culture in serum-free medium. The yield of Nb expressed in a shake flask is as high as 100 mg/L; compared with the stable expression produced by CHO cells, HEK-293 cells can usually be transiently transfected and expressed, providing some laboratory expression parameters, but these parameters need to be identified in the early stage, and are more suitable for preliminary expression conditions in the case of less laboratory costs. Mammalian cells that can effectively promote correct post-translational modifications are the most widely used expression system for antibody expression.

I. Nanobodies Antibody

Camel single-domain antibodies (sabs, VHHs or Nanobodies) are antibody fragments consisting only of heavy chain variable domains. These VHHs have unique structural and functional characteristics because of their small size, thermal stability and high solubility. Compared with traditional antibodies, VHHs can be manufactured in microorganisms, greatly saving costs, labor and time because VHHs lack the Fc domain containing N-linked oligosaccharides. So far, VHHs have been expressed in several production systems, from prokaryotes, yeast, fungi, insect cells, mammalian cell lines to plants.

不同形式的抗体-泰克生物.png

Figure 1 Different forms of antibodies

II. Nanoantibody Mammalian Cell Expression

So far, mammalian cells are the best expression system for producing functional antibodies, and most therapeutic antibodies can be expressed in this system. Among them, CHO cells are the most popular host because they grow rapidly in serum-free medium and adapt to suspension culture; in addition, they also express up to 100mg/L VHHs in shake flasks. Compared with stable expression produced in CHO cells, transient and semi-stable expression, which is usually produced in human embryonic kidney (HEK) 293 cells, is more suitable for laboratory environments because they require less time and effort, and they can also provide early indications of some key parameters that need to be identified early. However, HEK cells have not been used to produce VHHs so far, and there are few reports of antibody fragments produced using this method. Other mammalian cells, such as mouse B cells, have also been used to express VHHs. However, the production costs associated with mammalian cell lines are much higher than those associated with any other laboratory research platform.

Fusions consisting of VHHs and the Fc domain of human or mouse IgG show complement-dependent cytotoxicity (CDC) and inhibition of tumor cell activity; they may also extend the half-life of VHHs. Therefore, mammalian cells that effectively promote correct post-translational modifications are the most widely used platform for fusion expression.

III. Expression of nanobodies in different hosts

Host	Expression vector	VHH Format	Antigen	Production System	Yield
NSO myeloma	pSV2-neo	VHH-Fc	HEL	Petri dishes	~10ug/mL
CHO	pTT30	VHH-Fc	EGFR	10 mL of fresh CD DG44 media in125-mL shake flask in batchmode	100mg/L

TekBiotech uses the pIII protein of M13 bacteriophage for VHH antibody (nanobody) surface display. Our scientists constructed a phage vector with dual tags of Flag and 6*His tag, and prepared TG1 and XL1-Blue host bacteria with a transfection efficiency of 10^8. After 2-3 transfections and amplifications, we can provide customers with a high-quality VHH antibody display library with an effective library capacity of 10^8-10^9 and a packaging capacity of 10^13-10^16/ml particles.

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