ChromaX Chromatography Resins from BioLink for Efficient IgM Purification

IgM antibody is a type of immunoglobulin(Ig) that mainly exists in the early stage of humoral immune response. Looking through the evolution of immunoglobulins, IgM is one of the oldest types of antibodies. In the immune response process of host defense against pathogen invasion, each type of antibody has its unique functional effects. IgM is initially expressed on the surface of B lymphocytes and participates in the formation of the B-cell receptor (BCR). After activation of B cells, secretory IgM (sIgM) can be produced by plasma cells, participating in the early humoral immune response. Compared to other types of immunoglobulins, IgM has characteristics such as large molecular weight, multiple antigen binding sites, and high affinity, allowing it to play an important role in anti-infection immunity. However, in practical applications, purification of IgM is difficult due to its cross-reactivity with other immunoglobulins.

How to Obtain High Purity IgM Antibody?

Whether used in early diagnosis of pathogen infection or disease treatment, how to effectively obtain high purity IgM antibody is a problem we must solve. Compared to IgG, IgM has a larger molecular weight, lower solubility, and is more prone to degradation. Salt concentration, pH, and temperature will greatly affect the stability of IgM. Therefore, based on following the CIPP purification strategy, more considerations are needed for the purification of IgM. BioLink possesses a variety of chromatography resins that can meet purification requirements in different situations.

Method One: Affinity Chromatography

The Fc fragment of IgM has a large steric hindrance, so it is generally considered difficult to use Protein A for the affinity capture of IgM, but this is not absolute. In addition to binding to the antibody Fc segment, Protein A can also bind to the heavy chain variable region sequence encoded by the VH3 gene to capture the antibody. Therefore, for the purification of IgM, high-capacity, high alkali-resistant Protein A antibody affinity resins represented by BioLink's MaXtar® ARPA are still a worth-trying choice.

In cases where Protein A is not ideal, other structural domains of the Fab section can be considered for affinity resin selection. Among them, MaXtar® Protein L has a broad spectrum of antibody binding activity and can also be used for the purification of natural or recombinant IgM.

Considering the stability of IgM, the elution conditions of Protein A/L at low pH may be too harsh for some IgM, in which case other types of affinity chromatographic medium can be selected. Due to the large number of disulfide bonds in IgM, Bio-Link's MaXtar® PlasmidCap HR affinity resin based on the thiophilic affinity principle is also very suitable for the purification of IgM.

In addition to finished affinity resins, it is also possible to purify IgM antibodies by preparing specific antigens or antibodies, fixing them on pre-activated resins, and utilizing the specificity of IgM antibody binding to other immunoglobulins for purification. This method has high specificity but is more complex in operation, requiring the preparation of specific chromatography columns.

➤  BioLink MaXtar® ARPA antibody affinity resin is an affinity chromatography medium used for antibody purification through specific interaction between antigen and antibody. MaXtar® ARPA features high capacity, high flow rate, and alkali resistance: (1) High flow rate and high dynamic binding capacity shorten the processing time. (2) Modified alkali-resistant rProtein A ligand can withstand 0.5M NaOH for CIP.

Chromatography resin family photo

Method Two: Size Exclusion Chromatography (Including Composite Multi-Mode)

Considering the large molecular weight of IgM, the method of utilizing the difference in molecular size between IgM and other proteins to separate IgM from a mixture through BioLink's Chromstar® 6FF size exclusion resin is simple and easy to operate, but has low specificity and is prone to interference from large molecular impurities. Moreover, traditional size exclusion is not conducive to maintaining the stability of IgM due to reasons such as low sample intake, low flow rate, and the time-consuming process of chromatography. Therefore, the BioLink MaXtar® COLL multimodal resin series based on the size exclusion principle is also suitable for separating IgM from a mixture.

In contrast to traditional chromatography medium, BioLink's MaXtar® COLL multimodal resin is composed of two layers with different structures: the shell is a porous passive layer that ensures that large molecules above 400/700KD do not enter the pores but flow directly through the external water flow; the core is a spherical inner core coupled with both hydrophobic and adsorptive negatively charged groups to maximally bind host proteins, nucleic acids, and other impurities. This chromatography medium is ideal for the separation and purification of various large biological molecules. After cleaning and regeneration, the resin can be reused. Compared to traditional size exclusion, MaXtar® COLL 700 and MaXtar® COLL 400 under flow-through mode can support larger sample volumes and higher flow rates, resulting in efficiency increasing by a factor of 100,  making them more suitable for IgM purification.

➤BioLink Chromstar® 6FF size exclusion resin is a highly cross-linked  (with a 6% cross-linking ratio) size exclusion chromatography medium based on an improved agarose matrix. This resin aims to separate molecules based on differences in molecular weight and conformation. This chromatographic medium is ideal for the purification of various biological molecules, such as recombinant proteins, antibodies, nucleic acids, viruses and virus-like particles, polysaccharides, etc.

➤  BioLink MaXtar® COLL  is a multimodal chromatography resin. Unlike traditional chromatographic medium, it consists of two layers with different structures: the shell is a porous passive layer that ensures that large molecules above 400/700KD do not enter the pores but flow directly through the external water flow; the core is a spherical inner core coupled with both hydrophobic and adsorptive negatively charged groups to maximally bind host proteins, nucleic acids, and other impurities. This chromatographic medium is ideal for the separation and purification of various large biological samples.

Method Three: Ion Exchange Chromatography

Ion exchange chromatography is a technique that leverages the differences in ion strength and pH values between IgM and other proteins to separate IgM from a mixture through ion exchange resins. This method is simple to operate and effectively removes HCP, HCD, endotoxins, and viruses from the sample. The isoelectric point distribution of IgM typically ranges from 5.5 to 7.4, allowing for flexible application of either flow-through or elution modes based on the actual isoelectric points of different IgM molecules.

➤ BioLink's MaXtar® Q HR is a high-resolution strong anion exchange chromatography medium, utilizing the differences in charge properties and amounts of different molecules under specific conditions for separation. This chromatography medium is well-suited for the separation and purification of various biomolecules, including recombinant proteins, antibodies, nucleic acids, viruses and virus-like particles, polysaccharides, etc.

➤ BioLink's MaXtar® DEAE is a weak anion exchange chromatography medium, exploiting the differences in charge properties and amounts of different molecules under specific conditions for separation. This chromatography medium is effective for the separation and purification of various biomolecules. Compared to traditional weak anion exchange chromatography media, MaXtar® DEAE exhibits superior performance: (1) The improved MaXtar® framework provides greater rigidity, allowing for higher process flow rates under lower back pressure, enhancing process efficiency. (2) Upgraded ligand coupling improves the dynamic binding capacity of the chromatography medium.

➤ BioLink's MaXtar® MMA HR is an improved agarose-based multimodal chromatography medium. Unlike traditional ion exchange chromatography media, it consists of multiple different functional groups: amino, hydrogen bonding, and hydrophobic groups. This chromatography medium can remove major impurities such as nucleic acids, host proteins, aggregates, and viruses, making it widely applicable in the fields of polysaccharides, antibodies, virus carriers, or vaccines. Compared to traditional single-mode chromatography media, MaXtar® MMA HR demonstrates superior performance: (1) High dynamic binding capacity, capturing most impurities in a single step, saving time and costs. (2) The improved MaXtar® framework provides greater rigidity, allowing for higher process flow rates under lower back pressure, enhancing process efficiency. (3) Fine particle size design for high resolution. (4) Tolerant to a wide range of pH and conductivity, conducive to the exploration of optimal process conditions.

Table: IgM purification selection summary