search.noResults

search.searching

saml.title
dataCollection.invalidEmail
note.createNoteMessage

search.noResults

search.searching

orderForm.title

orderForm.productCode
orderForm.description
orderForm.quantity
orderForm.itemPrice
orderForm.price
orderForm.totalPrice
orderForm.deliveryDetails.billingAddress
orderForm.deliveryDetails.deliveryAddress
orderForm.noItems
18 May / June 2021


from universities and biopharmaceutical companies have been presented in recent webinars and conferences to illustrate the strength and application of iCIEF coupled to MS for mapping the main degradation pathways and critical quality attributes of biopharmaceuticals [13,14].


Conclusion


Highly efficient and robust iCIEF have been utilised in biopharma for routine quality and quantify protein charge heterogeneity at any point in biotherapeutic discovery, development, and production. The development and commercialisation of CDTT iCIEF technology described and demonstrated that automatic charge variant fraction collection based on iCIEF profile can be achieved with prep iCIEF in 40 min. The purity of the collected protein variant can be confirmed and used for further intact MS, peptide mapping, and SPR characterisation. The developed iCIEF-MS has been introduced. It has been demonstrated that a highly effective iCIEF-MS of protein charge variant can be realised in 15 min with conventional low flow ESI MS.


References 1. G. Rozing. Chromatograpy Today. (2019), 8-14.


2. Z. Sosic, D. Houde, A. Blum, T. Carlage, and Y. Lyubarskaya. Electrophoresis. 29 (2008), 4368-76.


Figure 7: Deconvolution of charge variants of NISTmAb.


charge variant and matrix are diluted about 100 times with the MS compatible solution before the charge variant entered the ESI source. This greatly reduces the risk of ESI source contamination with components in the matrix and effectively prevented potential capillary clogging.


Clean iCIEF-MS signals for all the charge variants were obtained. The top diagram in Figure 6 displayed the iCIEF profile and TIC profile of the iCIEF-MS of NISTmAb. Deconvolution of each charge variant mass spectrum generated multiple masses, corresponding to neutral glycosylation variants’ expected presence. The averaged and deconvoluted mass spectra of each charge variant peak from the iCIEF-MS analysis of intact NISTmAb sample are shown in Figure 7. Overall, the deconvoluted mass spectra’s peak shapes show good peak symmetry, indicating minimal interference from the carrier ampholytes adducts. The identity of each charge variant species was based on the known molecular weight of the main peak


and Δmass relative to it. The two basic peaks, basic 1 and basic 2, can be readily assigned as one unprocessed Lys residue at the heavy chain C-terminus and two unprocessed Lys residues at the heavy chain C-terminus, respectively. The deconvoluted mass spectrum of acidic peak 1 showed inconsistent Δmass to that of the main peak. Different Post translational modifications (PTMs) such as deamidation, glycation, cysteinylation, glutathionylation might be expected. Unambiguous identification of these PTMs can be done with other techniques such as iCIEF fraction collection, controlled enzyme digestion, deglycosylation.


Streamlining iCIEF-MS of CDTT iCIEF and Thermo Fisher Scientific’s powerful Orbitrap MS greatly facilitates protein charge variant characterisation. Unattended iCIEF-MS has been enabled with contact closure between the CEInfinite CE system and QEplus MS, providing high resolution and high-efficiency iCIEF-MS for routine characterisation of biopharmaceuticals and other macromolecules. Case studies


3. D. A. Michels, et al. BioProcess International. 9 (2011), 48-54. 4. T. Huang. US Patent # 10935519. 5. R. S. Rogers et al. The AAPS Journal. 20 (2018), 7.


6. C. Montealegre, C. Neusüß. Electrophoresis 39(2018), 1151-1154.


7. Y. Yan, A.P. Liu, S.Wang, T.J. Daly, and N. Li. Anal. Chem. 90(2018), 13013-13020.


8. J.Dai, J. Lamp, Q. Xia, Y. Zhang. Anal. Chem. 90(2018), 2246–2254.


9. J. Dai, Y. Zhang, Anal. Chem. 90(2018),14527-14534.


10. L.Wang, and D.D.Y. Chen. 40(2019), ELECTROPHORESIS. 2899-2907.


11. S. Mack et al. ELECTROPHORESIS. 40(2019), 3084-3091.


.12 T. Huang, XZ. Wu, J. Pawliszyn, Anal. Chem. 72(2000), 4758-4761.


13. C. Neusüß, C. Sönksen, T. Huang. https:// www.selectscience.net/expert-insight/the- future-of-cief-mass-spec-techniques-for-protein- characterization/?artID=49878.


14. D.B. Kristensen. Presented at Bioprocessing Summit Europe 2021.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60