Imaged Capillary Iso-Electric Focussing: Background, Status, and Perspectives

8 February / March 2019 Imaged Capillary Iso-Electric Focussing: Background, Status, and Perspectives by Gerard Rozing, ROZING.COM Consulting, Karlsruhe, Germany Iso-electric focusing (IEF) has proven to be a very versatile separation method for the characterisation, identification, purity determination, and quantitation of amphoteric substances such as peptides and proteins. It is an indispensable and invaluable analytical tool in proteomics research, in the development of new biotherapeutics and in quality- and process-control in their manufacturing. The method is based on arranging and separating proteins according to their isoelectric points or pI, allowing to establish changes in the charge state of the molecules after structural modifications such as deamidation, oxidation, isomerisation, N-terminus modification, disulfide formation, and glycosylation. Modifications that change biochemical characteristics of biotherapeutics like their isoelectric point (pI), reduce their safety and long-term stability, influence drug efficacy, and increase undesired immunogenicity. Nowadays, iso-electric focusing is molecules on an IPG are static, better application is a discrete independent step performed in two different formats: reproducibility is obtained. These IPGs were after the pH gradient has been established, introduced as a commercial product by LKB which in principle can take place at any of Sweden (now GE Healthcare). The IPGs physical location along the gradient on Slab Gel IEF are prepared on small plates or strips the the gel plate or over the length of an IPG latter of which is used as the first dimension strip. Conventional CE sample introduction Early iso-electric focusing was executed in 2D Gel Electrophoresis. occurring at the inlet end of the separation on a flat substrate (glass or plastic plate) capillary after the pH gradient has been coated with a mixture of polyacrylamide formed is not meaningful in CIEF. or agarose to suppress hydrodynamic or Capillary Iso-Electric electro-osmotic flow and so-called carrier Hjertén was the first to describe IEF in a ampholytes to establish a pH gradient. Focusing (CIEF) CE capillary [2] in 1985 followed by many Carrier ampholytes (CA, amphoteric Despite the improvement of IEF by using practical and theoretical reports in peer electrolytes) are oligomeric molecules with prefab IPG gel plates, the method remained reviewed journals and textbooks [3,4,5,6] . MW between 300-1000 obtained by the laborious, lacked automation, on-line Despite the significant improvements in the reaction of polyamines with acrylic acid (or detection with electronic data processing methodology on CE-instrumentation, CIEF similar structured unsaturated sulphonic and documentation. The introduction of still requires significant user experience acids). Each carrier ampholyte molecule, instrumentation for Capillary Electrophoresis and expertise. The work by Mack et al [7], therefore, will contain several ionisable (CE) followed quickly, employing IEF in a describing a systematic study on the usage acidic and basic groups which determine the capillary. However, IEF, as described before, of so-called blocking agents that confined pI of each ampholyte molecule. A mixture of is a spatial separation method which takes the focussing to occur in front of the PoD, structurally different CA’s will cover a range some time for focusing to complete and has been a major step forward in improving of pI’s. The plate is flanked with trenches sample molecules have congregated the method. containing a low pH solution (anolyte) and a as separate bands or spots at a specific high pH solution (catholyte). location. This in contrast to CE, which is a temporal separation method, in which the Principle of CIEF [8] Laborious gel preparation with ampholyte, time is measured from the moment t0 when precise sample application, the visualisation It is appropriate to first look into details the sample molecules enter the capillary of the separated bands, and the unstable pH of the CIEF process to appreciate the until a time t when a compound passes the gradient made this IEF method moderately i improvement with imaged CIEF. point of detection (PoD). reproducible and presented the user with For CIEF a coated fused silica (FS) capillary ambiguous separation interpretations. The biggest challenge in implementing is used, typically 50 µm i.d. and 30-40 IEF in a capillary, is to focus all the sample A significant improvement of the IEF method cm depending on the brand of the CE molecules in front of the point of detection was obtained with the first description of instrument used. The internal capillary during the focusing stage and once focusing Immobilised pH Gradient (IPG) gels by coating (fluorocarbon, polyvinyl alcohol, has been completed, to move the focussed Bjellqvist et al [1] which makes the polymer DB-WAX) suppresses the electro endo- zones past the PoD. In addition, one must gel itself amphoteric. Since the ampholyte osmotic flow (EOF). At the beginning of a realise, that with IPG gels, the sample 9 capillary. When the voltage is switched on the situation changes as depicted in Figure 2. When the focussing process starts, highly mobile hydrogen ions move towards the cathode, whilst hydroxyl ions move to the Figure 1: Depiction of the capillary composition at the beginning of a CE run. The proteins to be anode. At the same time low pI molecules separated have pI 5 and 7. Carrier ampholytes, pI markers and additives are homogenously distributed which are initially positively charged move over the capillary and not visualised here. (picture with permission of Agilent Technologies) towards the cathode and negatively charged high pI molecules move to the anode. Upon migration to the electrode of opposite charge, an amphoteric molecule is protonated/deprotonated and focuses in a small zone. This is a dynamic process, with the amount of protonation and deprotonation depending on the pKa and pKb of the ionisable groups in each analyte molecule. The completion of the focusing process in a CE instrument is monitored by the current. When all molecules are neutralised and focused, the current will have dropped significantly to a minimum level. This Figure 2: Schematic of initial steps of the focussing process. (Picture with permission of Agilent normally occurs after approx. 20-30 minutes. Technologies) At this time, it is believed that all proteins are arranged along the capillary according CIEF run, the whole capillary is filled with solution is determined by the concentration to their pI. a mixture containing, carrier ampholytes, and dissociation state of all substances covering a specific pI range e.g. 3-8, present. The inlet vial is filled with a low Figure 2 shows the problem with this approach. The PoD is remote (7-10 cm blocking agents, the protein or peptide pH solution (anolyte, H3PO4) and a positive sample to be analysed, and pI markers. voltage is applied here, resulting in this depending on the CE-instrument) from Additives may be added like methylcellulose electrode becoming the anode. The outlet the end of the capillary. Therefore, high pI to suppress residual EOF or urea, to improve vial contains a high pH (catholyte, NaOH) proteins may become focused outside of the solubility of the focused molecules. The solution, is grounded and the electrode at the PoD and will be lost at the start of the absence of EOF is essential in the execution this point becomes the cathode. mobilisation. of CIEF since the focussing process is This is illustrated in Figures 1 and 2. In a collaboration with scientists from disturbed by flow in the capillary. Beckman Coulter, Vigh developed a solution Before the focussing process commences [7]. So-called sacrificing ampholytes or All substances are distributed molecules with a low pI will have a positive blocking agents, which have a pI lower or homogeneously over the whole capillary charge while molecules with high pI will higher than the pI of all sample proteins at the start. The overall pH of the sample have a negative charge at any location in the and pI markers, are introduced into the sample mixture. Iminodiacetic acid, pI 2.2 and arginine, pI 10.7 are examples. Now it must be realised that the length of a zone occupied by any ampholyte in the capillary depends on its pI, its amount in the capillary as well as the pI values and amounts of all other ampholytes in the capillary. When the concentration of the blocker is chosen properly, the focussing of analyte molecules and pI markers occurs before the point of detection. The high pI blocking agent will focus beyond the PoD. An illustration is given in Figure 3 taken from a technical note [9]. The cathodic peaks are caused by the movement of high pI sample components from the cathodic side of the capillary which Figure 3: cIEF separation of peptide pI markers (Table 3). Sample: 200 μL of cIEF gel, 12.0 μL of Pharmalyte passes the PoD during the focusing phase 3-10, 2.0 μL of each pI marker (1.25 mM), 18.0 μL of cathodic stabiliser (0.5 M Arg.) and 4.0 μL of anodic sta- towards the anodic side. They focus before biliser (0.2 M IDA). Method: focusing: 15 min at 25 kV; chemical mobilisation: 25 min at 30 kV. Anolyte: 200 the PoD. mM phosphoric acid. Catholyte: 300 mM sodium hydroxide. Chemical mobilising solution: 350 mM acetic acid. (Figure with permission from Sciex Separations) 10 February / March 2019 [10,11,12] . In their original work, a short, 4 cm, glass separation capillary, was used and like CIEF was connected to the anolyte and catholyte vial respectively. In a later implementation, the separation capillary was connected to an inlet and outlet capillary by a hollow fibre membrane which allows the passage of protons and hydroxyl anions into the separation capillary. This method was commercialised by Convergent Biosciences (now Protein Simple) who entered the market in 2000 with the ICE280 system, followed by Advanced Electrophoresis Solutions with their CEInfinite system in 2016.

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