Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases

Combining multidimensional liquid chromatography and MALDI–TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer

“…On the other hand, pH gradient IEC is a powerful technique for the separation of proteins having close charge properties; therefore pH gradient has been usually employed for separating proteins with close isoelectric point (pI) values [78]. In pH gradient IEC proteins usually elute in order of their pI [78][79][80], and this order is frequently not preserved using salt gradient IEC, thus pH gradient IEC should be more suitable than salt gradient IEC for separating distinct structural classes of toxins having isoelectric point differences. Moreover pH gradient fractionation can be used as an analytical tool for design and pH optimization of salt gradient ion-exchange chromatography; and both modes can be combined for fractionation of crude samples [78,81].…”

“…Salt gradient IEC is widely employed in protein purification due to its ease of use, loading capacity, versatility and high resolution. However, proteins with the same number of effective charges are released from the ion exchanger close to each other, thus limiting the selectivity of salt gradient IEC when complex mixtures are separated [79]. On the other hand, pH gradient IEC is a powerful technique for the separation of proteins having close charge properties; therefore pH gradient has been usually employed for separating proteins with close isoelectric point (pI) values [78].…”

Modeling of dual gradient elution in ion exchange and mixed-mode chromatography

“…This can be achieved by carefully selecting buffer components which provide stable buffering capacity over a wide pH range (e.g. pH [4][5][6][7][8][9][10][11], preferably with low ionic strengths and which do not interact with the stationary phase [10].…”

“…Alternatively, pH gradient elution is becoming popular in analytical and preparative chromatographic processes not only for mAb isoforms separation but also for closely related protein variants separation [8][9][10][11].…”

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

“…31 Their system overcomes the previously described need to compensate gradient non-linearities with a software-enabled, algorithmic control of the gradient mixing, 32 making it simpler and easier to use and implement. Here, we describe the use of this buffer system for the purification of BsAb heterodimers from homodimers on a preparative scale.…”