gM purification is no longer difficult, super porous media to help

IgM antibody, which has a pioneering immune function, is widely used in the prevention, early diagnosis and treatment of disease infection. However, obtaining high purity IgM antibodies was the first problem we had to solve. The chromatographic medium with the advantage of macropores is a worthy choice for purifying IgM, which has the title of "macroglobulin".

 Unique Features of Macroglobulin IgM

Immunoglobulins are classified into five classes according to the antigenicity of their heavy chains: IgG, IgM, IgA, IgD and IgE. IgM is by far the largest antibody actually found in the human circulatory system, usually in the form of pentamers or hexamers, with molecular weights of about 900 kDa or 1050 kDa, respectively. Its advantages are as follows:

Because IgM antibodies exist in blood and lymph, they are usually the first antibodies that appear first after infection. IgM antibodies can play a pioneering role in immunity after generation, and can activate complement and improve autoimmune conditioning. It also has a certain role in clearing viruses and microorganisms in the body.

IgM has 10 arms that bind to the target antigen with higher affinity than IgG has 2 arms.

Relevant data show that IgM may be more effective than IgG in diagnosing autoimmune diseases and some cancers, and foreign companies have devoted themselves to converting IgG into IgM structures to improve affinity to create bi-specific antibodies.

IgM purification is difficult

IgM antibody is more difficult to purify than mature IgG antibody.

Affinity chromatography is the best purification method for antibody purification, but Protein L with broad spectrum Ig binding ability can be used for human IgM, while IgM from other species has no binding site with Protein L, so it is less commercialized and costly.

At present, there are purification methods on the market, including ammonium acid precipitation, gel filtration, affinity chromatography, ion exchange, etc. Most of these methods use common chromatographic media, more or less have some limitations. Due to the large molecular weight of IgM, the pore size of ordinary agarose microspheres and polymer microspheres is small, the antibody cannot enter the interior of the microsphere channel, resulting in extremely low binding capacity. Meanwhile, the narrow pore size will lead to the antibody passing through in a diffusion manner, with slow mass penetration rate, low throughput, long separation time, and difficulty in maintaining the stability of IgM. Moreover, the pressure resistance of ordinary agarose microspheres and polymer microspheres is limited, which is not conducive to high column efficiency.

Therefore, more comprehensive consideration is needed in the process of purifying IgM.

IgM purification is no longer difficult

Products that can solve difficulties and pain points are the biggest demand in the market. Kono focused on the current difficulties and pain points of IgM purification, constantly explored, combined with its own ultra-large pore technology, developed a unique way to launch an efficient IgM purification solution.

Scheme 1: Affinity Method

Step ① Affinity chromatography purification:

IgM was captured using Protein L Sup-porousd ultra-large pore affinity chromatography medium.

Step 2 Ion exchange chromatography purification:

Using MoSphere? 50V Q super pore anion exchange chromatography medium, adsorption IgM, removal of trace impurities.

Option 2: Exclusion

Step ① Compound mode chromatography purification:

Using AdMix Chromrose? FF (anion exchange and hydrophobic dual function) complex mode chromatography medium, adsorption host cell impurities, IgM molecular weight, exclusion direct flow through.

Step 2 Ion exchange chromatography purification:

Using MoSphere? 50V Q super pore anion exchange chromatography medium, adsorption IgM.

Of course, IgM antibody purification scheme is not only the above two kinds, downstream purification is a complicated process, according to local conditions according to the fermentation broth to adjust the purification scheme is the most wise choice. Affinity method and exclusion method are suitable for recombinant IgM antibody. For ascitic fluid and serum antibody, due to the large number of antibody subtypes, it is necessary to design specific purification schemes. Compared with imported standardized products, Kono is more inclined to flexibly customize products and solutions according to market needs.

Multi-aperture design for different application scenarios

The interaction between ligand and target substance takes place in the interior of chromatographic medium particles. Accurate pore size design is very important for purification. According to different application scenarios of customers, Konosay has developed macroporous polymer ion exchange chromatography media with different particle sizes and pore sizes to meet the application needs of users.

The advantages of Konosay macroporous ion exchange chromatography media:

High protein activity:

The design of super-large pore size can obviously reduce the unfolding degree of protein and improve the activity of protein.

Low non-specific adsorption:

After hydrophilic modification, ion exchange groups are bonded to the surface of microspheres, resulting in low non-specific adsorption and high mechanization.

High flow rate, high load capacity:

Ultra-large pore size has better permeability, faster molecular mass transfer rate, and can maintain higher loading capacity at high flow rate, and the loading capacity is increased by more than 10 times.

High stability, high recovery:

Super-large pore size significantly improves the thermal stability of biomolecules adsorbed on chromatographic media and has higher yield.

The only one in China

Imported brands monopolized the chromatographic filler market for decades, and the epidemic gave domestic brands an opportunity. Kono Sai was born at the height of the epidemic. The team members did not have "Dr. Overseas Returnees","American Green Card" or "European and American Permanent Residency". It was an authentic "country bumpkin" team. Based on the enthusiasm for the industry and the deep understanding of the market, this "ground beetle" team starts from "making products that others have", deeply explores market demand, and gradually "makes products that others do not have" according to market demand.

Plasmid, AAV virus vector, LV virus vector, oncolytic virus, influenza virus, IgM antibody, IgA antibody, mRNA vaccine, polysaccharide conjugate vaccine and other biomolecules have not been well purified because of their huge volume. Kono's team has developed a new ultra-large pore chromatography medium for this market pain point.

Super-large pore ball technology has long been established by scientists theoretical basis, but because of the "hole big fragile ball" reason has not been commercially produced.

Since its establishment, Kono Biotech has always adhered to the R & D service policy of "customer-centered and market-oriented". It has been constantly looking for the best solution for the difficult pain points of customers in the purification stage. Through unremitting efforts in the past three years, it has worked hard to tackle key problems, refined and polished continuously, and finally broke through the technical bottleneck to achieve success. It is currently the only enterprise in China that has mastered the technology of super-large hole ball making and successfully practiced it.

In the next step, Kono Sai will continue to develop ultra-large pore affinity products, mRNA affinity, plasmid affinity, AAV affinity and exosome affinity by using ultra-large pore technology, continuously supplement the shortcomings of domestic chromatography, make products needed by the market, and better meet the purification needs of domestic biological pharmaceutical enterprises.