Purification Techniques | A Tenfold Increase in Purification Efficiency for High Molecular Weight Macromolecules: A Remarkable Breakthrough

Macromolecules, including viruses, VLPs (virus-like particles), viral vectors, and IgM antibodies, typically possess molecular weights surpassing several hundred kilodaltons (kDa).

In both scientific research and the pharmaceutical industry, obtaining an adequate quantity of high-purity samples is essential. Taking soluble samples as an example, commonly employed purification methods include centrifugation, membrane filtration, and chromatography. Among these methods, protein chromatography stands out as the technique with the highest resolution and the most diverse separation principles. Based on different chromatographic principles, various modes of chromatography are utilized, including gel filtration chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and multimodal chromatography. By selecting the appropriate chromatography resin based on the key differences between the target molecule and impurities, one can achieve significant efficiency and effectiveness in the purification process.

Taking the cultivation of 293T cells as an example, the molecular weights of most host proteins and host nucleic acids, which serve as impurities, are below 100 kDa. Therefore, there is a significant molecular weight difference between these impurities and viruses. Gel filtration chromatography resins, such as BioLink's Chromstar 4FF, can be used for separation purposes. In this process, viruses elute outside the void volume while impurities elute within the void volume, resulting in a perfect separation.

Figure 2: Chromatogram of Influenza Virus Purification using Chromstar 4FF

Sample: Influenza virus stock solution

Column volume: 150ml

Sample volume: 10ml

Flow rate: 2ml/min

Due to the typical sample loading volume of 5% to 15% of the column volume for gel filtration chromatography resin. To increase the processing capacity, it is common to use a higher column bed height, which results in a larger column bed volume.

The above approach is a good one.

But here's a better option for you: MaXtar Series.

MaXtar COLL series produced by BioLink is a multimodal chromatography resin specifically designed for large macromolecules. It includes two variants, MaXtar COLL 700 and MaXtar COLL 400, which are suitable for macromolecules with molecular weights exceeding 700 kDa and 400 kDa, respectively.

Figure 3: Chromatogram of VLP Purification using MaXtar COLL 400

Sample: VLP (monomer 60 kDa, 120-mers)

Column volume: 30 mL

Sample loading volume: 300 mL

Flow rate: 7 mL/min

The COLL series multimodal chromatography resin offers the unique advantage of accommodating sample loading volumes up to 5-10 times the column volume, allowing for increased sample capacity. Moreover, the flow rate can be enhanced by 3-5 times, demonstrating its impressive efficiency. It should be noted that to fully exploit the diverse ligand binding properties within the COLL series, it is recommended to supplement the buffer solution with 100-150 mM NaCl. During the CIP cleaning step, it is advisable to utilize a solution of 1M NaOH and 30% isopropanol to ensure effective cleaning.

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