Using nuclear magnetic resonance (NMR) technology reveals the outline of protein

Monoclonal antibodies (MAbs) can be used to identify nearly all of the antigen, including all the cells in the human body surface and soluble receptors.As a result, their development is one of the most active areas in pharmaceutical research, so we will seek to use the great potential of these molecules.

Accurate characterization of the protein therapeutic agent (mAbs), senior structure (HOS) is crucial, because their structure and their functions highly correlated.In addition, in the manufacturing process of HOS may be different due to different batches or use of the manufacturing process, which means that the HOS monitoring for the quality control is also very important.

Typically, scientists using two-dimensional nuclear magnetic resonance (NMR) to represent three dimensional structure of protein, or "fingerprint (map).MAbs is so much larger, however, there are many overlapping spectra produced in this way, it's hard to explain.In addition, also can change the protein glycosylation HOS, but in general, describe the characteristics of glycosylation need further steps, such as protein enzyme solution and liquid chromatography - mass spectrometry.

In a 2015 study, Leszek Poppe led the researchers suggest that a virus called PROFILE (protein fingerprint by the line shape enhancement), namely through the line shape of enhanced protein fingerprint of one-dimensional nuclear magnetic resonance (NMR) technology can be simplified, speed up and increase the MAb characterization of details.

The team first studied six batches of glycoprotein agents, called Epoetin alfa, they are made up of two different processes (and rhEPO rhEPO - A - B) manufacturing.The researchers analyzed the protein of the native, folding status to reflect their HOS, and thermal expansion state, this reflects the amino acids in the protein sequence.

Using brooke Avance nuclear magnetic resonance spectrometer, combined with low temperature probe and brooke three resonance automatic sampler, the researchers used the PROFILE method of similarity analysis.The team found that the native rhEPO - A and rhEPO - and thermal expansion of rhEPO - A and B rhEPO - B is almost the same similarities between each other.This suggests that the rhEPO - the difference between A and rhEPO - B is A structural change, cannot be attributed to the HOS.

But the researchers found that when using the method of analyzing IgG1 antibodies the opposite result.Here, their commitment to the antibodies in the native and not folded state of glycosylation and analyzes similarities to glycosylation forms.It shows that the native protein glycosylation and significant difference between glycosylation forms, but not folded protein glycosylation and there is no difference between glycosylation forms.The only explanation for these findings, the researchers say, is to glycosylation of IgG1 cause HOS changed much of the protein.

The research team also showed that the PROFILE technology selectivity is much larger than 2 d NMR.Use IgG1 and IgG2 antibodies, they found that the PROFILE could be detected 68% of the differences between the two proteins HOS and 2 d NMR can detect 42% difference.Compared with the two-dimensional method, molecular samples of low concentration PROFILE method to differentiate IgG1 IgG2 molecules are more effective.

The researchers report in the Analytical Chemistry (Analytical Chemistry), published their findings, they said, so the PROFILE can provide more than 2 d HOS information.They point out that compared with the two-dimensional nuclear magnetic resonance (NMR), it also has other advantages, such as its isotope labeling or cracking, the samples don't need to and can provide faster acquisition time.They conclude, therefore, the method should be found in the biopharmaceutical development and quality control of a variety of purposes.

References:

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• Kotsovilis S & Andreakos e. Therapeutic human monoclonal antibodies in inflammatory diseases. Methods Mol Biol 2014;1060-37-59.
• Poppe L, Jordan JB, Lawson K, et al. The Profiling Formulated Monoclonal Antibodies by 1 h NMR Spectroscopy. Anal Chem, 2013;85:9623-9629.
• Poppe L, Jordan JB, Rogers G, et al., On the Analytical Superiority of 1 d NMR for Fingerprinting the who Order Structure of Protein Therapeutics Compared to Multidimensional NMR Methods. Anal Chem, 2015;87:5539-5545.
• Local H, Thomas D, Lopez a. Monoclonal Antibodies: Therapeutic Uses. ELS 2013;DOI: 10.1002/9780470015902. A0002176. Pub3