A high-throughput screening assay on a microfluidic chip was developed for the determination of charge variants of monocolonal antibodies (mAbs) in pI range of 7-10. This method utilizes microchip zone electrophoresis for rapid separation (<90 s) of mAb charge variants that are labeled fluorescently without altering the overall charge. The microfluidic assay achieves between 8- and 90-fold times faster separation time over conventional methods while maintaining comparable resolution and profiles of charge variant distributions. We further characterized the assay with respect to (i) the effect of pH on resolution, (ii) the effect of excipients and buffering agents, (iii) the performance of the assay compared to conventional methods, and (vi) the reproducibility of charge variant profiles. Finally, we explored the utility of the assay with four case studies: (i) monitoring C-terminal lysine modification of a mAb, (ii) quantifying the extent of deamidation of a mAb, (iii) providing charge variant information on which to base clone selection, and (iv) making process parameter-related decisions from a "design of experiment" (DoE) study. The results of these case studies demonstrate the applicability of the microfluidic assay for high-throughput monitoring of mAb quality in process development of biopharmaceuticals.

Rearrangements of 1-and 2-Cyano-2-norbornyl CationsSulfonates of exo-2-hydroxynorbornane-endo-2-carbonitrile (2c-e) were found to rearrange exothermally to give sulfonates of exo-2-hydroxynorbornane-1-carbonitrile (6c -e), solvolysis being a minor side reaction. In contrast, the analogous endo substrates (3c -e) afforded the rearranged alcohol 6a and tricyclo[2.2.1.02~6]heptane-l-carbonitrile (4) as the major products. We have not been able to trap 2-cyano-2-norbornyl cations by external nucleophiles or by internal 6,2-shifts of hydrogen or carbon. On the other hand, there is good evidence for the generation of 1-cyano-2-norbornyl cations from both 3d, e and 6e. The degenerate 6,2-H shift in these species has been uncovered by means of labeled or optically active precursors. The 1-CN substituent clearly promotes the 6,2-H shift relative to the parent 2-norbornyl cation, but is inferior to 1-CzFS. Ring expansion of a spiroannelated cyclopropane, involving a 6,2 shift of carbon, was also observed (25, 26 -+ 21, 28). Our data strongly suggest that 2-cyano-2-norbornyl cations are less stable than 1-cyano-2-norbornyl cations. The reaction rates of 2c versus 6c do not reflect the stability of the incipient carbocations, owing to the large difference in ground state energy.Destabilisierte Carbokationen, die elektronenziehende Substituenten am positiv geladenen C-Atom oder in dessen Nachbarschaft tragen, finden in neuerer Zeit starkes Interesse"]. Cyan-und Perfluoralkyl-Gruppen erweisen sich als besonders wirksam. In das umlagerungsfreudige und intensiv untersuchte Norbornyl-System wurde CN bereits in l -S t e l l~n g [ *~~~ und 6-Stell~ng[~.~' eingefiihrt. uber das Verhalten von 2-Cyan-2-norbornyl-Edukten war bisher nichts bekannt; diese Lucke wird nun geschlossen. Ferner berichten wir iiber 2,6-H-Verschiebungen in CN-substituierten Norbornyl-Kationen und uber analoge Kohlenstoff-Verschiebungen von Spiro(norbornan-2,1'-cyclopropyl-6-yl)-Kationen. Umlagerung und Solvolyse von Sulfonsaureestern des 2-Hydroxy-bicyclo[ 2.2.11 heptan-2-carbonitrilsDie Umsetzung von Norbornan-2-on (1) mit Cyanwasserstoff ist in der L i t e r a t~r~~, '~ erwahnt, ohne dal3 die epimeren Cyanhydrine 2a, 3a getrennt und charakterisiert wurden. Wie wir fanden, erhalt man aus 1 mit NaCN/H2S04 vorwiegend die exo-Verbindung 2 a (thermodynamische Kontrolle; 2a: 3a = 87: 13). Trimethylsilylcyanid/Zn12n liefert fast ausschlieBlich den endo-Silylether 3 b (kinetische Kontrolle; 3b:2b = 96:4), aus dem 3a durch saure Hydrolyse zuganglich ist. Die Stereoisomeren lieBen sich durch HPLC trennen bzw. reinigen. Die Konfigurationszuordnung beruht -neben der Darstellungsweise -auf den 'H-NMRSpektren. Die Signale von 3-H,,, und 3-Hend, (J3x,3n = 13.5 Hz) sind durch die Kopplungen J3xp bzw. J3n,7a leicht zu unterscheiden. Das Signal von 3-H,,, ist in 2a durch die vicinale OH-Gruppe hochfeldverschoben (6 = 1.77), in 3a durch die vicinale CN-Gruppe tieffeldverschoben (6 = 2.25).Die Signale von 3-Hend, verhalten sich umgekehrt (2a: 6 = 1.90; 3a: 6 = 1.35). 6 IAus 2 a...

Rearrangements of 1-and 2-Cyano-2-norbornyl Cations.-2-Carbonitriles of type (I) undergo Wagner-Meerwein rearrangement in dioxane/H2O to yield 1-carbonitriles like (II) and (III). Attempts to trap norbornyl cation intermediates fail, but degenerate 6,2-H shift in the labeled nitriles (IV) and (VII) as well as in optically active nitriles is good evidence for their generation. In spiroannelated cyclopropane analogues (cf. (VIII)), a 6,2-C shift can also be observed. -(GEIER, H.; KAUTZ, C. B.; KIRMSE, W.; LANDSCHEIDT, H.; SCHIMPF, I.; SIEGFRIED, R.; Chem.