In recent years, non-IgG antibodies exhibit certain therapeutic advantages over widely applied IgG monoclonal antibodies in tumors, infections, and inflammation. To further investigate and offer better services for non-IgG antibodies, Creative Biolabs has specially constructed a professional platform to provide non-IgG antibodies production and purification services for customers in need.
Introduction of IgM
IgM is the earliest antibody class expressed in the human fetus, and it is also the major isotype produced at the early stage in the primary response to antigens. In general, IgMs are produced by the immune system and play an important role in both innate and adaptive humoral immune responses. Just similar to other types of antibody molecules, IgM monomeric molecular consists of two identical heavy chains (μ) and two light chains (κ or λ). The predominant form of IgM in human serum is pentamer (~950 kDa) which is composed of five monomers connected with the joining-(J) chain. The other forms, such as hexamers (~1100kDa) and monomers (~170kDa), can also be found at rather a low abundance in vivo.
As an alternative therapeutic agent for IgG, IgM therapy benefits from the superior mobilization of effector functions. It has been found that IgM in pentameric form is the most efficient in the classical pathway of complement system activation. What’s more, the mucosal immunity can be attained by transfer of IgM through the epithelial barrier. Besides the immune protective effect, IgM antibody exhibits great therapeutic potential in the treatment of various tumors both in laboratory and clinical research. Therefore, a large amount of high-quality IgM is necessary for pre-clinical and clinical testing.
Fig. 1 Structure of (A) IgG;
(B) pentameric IgM; and (C) hexameric IgM. (Mader, 2013)
IgM Production
The main workhorses for recombinant antibody production are CHO cells and HEK293 cells. Creative Biolabs has successfully produced, purified and optimized a full range of IgM antibodies in a variety species by recombinant DNA technology based on our advanced Expression System Platform. Our recombinant IgM antibodies expression system extensively includes Chinese hamster ovary (CHO) cell lines, PER. C6 human cell line, myeloma cell lines, etc. Once the sequence is provided, we can start with gene synthesis, vector construction, and plasmid preparation. After the subsequent transient transfection and stable cell line culture, IgM antibody can be obtained as a pentamer or hexamer form linked by inter-molecule disulfide bonds and a J-chain. In addition, traditional hybridoma technology (both hetero-hybridomas and human-human hybridoma) also can be utilized for IgM production, which may involve a modification before direct cell fusion.
IgM Purification
Commonly used affinity chromatography is not applicable for the purification of IgM since that IgM can’t bind well to protein A/G. Furthermore, the narrow solubility and poor stability make it challenging for IgM purification. Large size property allows size exclusion chromatography applied for IgM purification with the limitation of rate-limiting. Ion exchange chromatography is a relatively suitable option for the purification of IgM antibody. The optional IgM purification methods include 3-step strategy (CHT/AIX/CIX), 2-step strategy (PEG/AIX), SXC, and capture select.
- For the 3-step strategy (CHT/AIX/CIX), we use ceramic hydroxya-patite (CHT) for a first enrichment step, followed by anion (AIX) and cation (CIX) exchange steps to get a 99% purity and 51%-80% recovery rate.
- For the 2-step strategy (PEG/AIX), we use polyethylene glycol (PEG) precipitation followed by AIX with nine different monoclonal IgM molecules from hybridoma culture supernatant to get a 96% purity and 28%-84% recovery rate.
- The novel size exclusion chromatography (SXC) method, whose principle is that retention of the target molecule to the hydrophilic matrix is achieved by steric exclusion of water by PEG, resulting in 90% purity and 90% recovery rate.
- The capture select method is achieved by the 14 kDa llama antibody fragments that recognize the human IgM μ-chain which are coupled to sepharose beads.
Fig.2 A process of our IgM production and purification.
Based on our comprehensive antibody platforms and professional experts, Creative Biolabs has absolute advantages in providing non-IgG antibodies production and purification services. Additionally, our ready-made/custom non-IgG antibody products and related services will help you achieve milestone development.
Just directly contact us if you are interested, our scientist will customize the most suitable solutions for your programs.
Reference
- Mader, A.; et al. Recombinant IgM expression in mammalian cells: A target protein challenging biotechnological production. Advances in Bioscience and Biotechnology. 2013, 4(4A): 38-43.
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!! For Research Use Only. Our products and services are NOT intended for diagnostic or therapeutic applications.
