Chromatography


Capillary Isoelectric Focusing – A Comparison of Different Carrier Ampholytes for Monoclonal Antibody Charge Heterogeneity Analysis


Christian Wenz, Agilent Technologies, Inc, Hewlett-Packard-Strasse 8, 76337 Waldbronn, Germany


This article will compare the performance of four commercially available brands of wide-range pH 3 to 10 carrier ampholytes (CA’s), Pharmalyte, Servalyt, HR, and SH AESlyte, for high-resolution capillary isoelectric focusing on fl uorocarbon coated capillaries. The carrier ampholytes-specifi c background was increased for Servalyt, and slightly increased for SH AESlyte in comparison to Pharmalyte and HR AESlyte. With all tested brands of carrier ampholytes, it was possible to analyse monoclonal antibody charge isoforms with high resolution and precision. The peak pattern of the test sample showing multiple isoform peaks in the pH range 6 to 7 was similar with Pharmalyte and HR AESlyte. A slightly increased resolution was observed with SH AESlyte, and the best resolution with Servalyt. The intermediate precision of experimentally determined isoelectric points obtained with three different capillary batches on three different days was better than 0.1 %RSD for all brands of carrier ampholytes and all isoforms. In terms of percent area, the observed intermediate precision was better than 3 %RSD for Pharmalyte and HR AESlyte, and better than 9 %RSD for SH AESlyte and Servalyt, with one exception.


Introduction


Capillary electrophoresis (CE) is a well-suited analytical tool for protein characterisation due to its simple instrumentation, superior separation effi ciency, small sample consumption, and short analysis time. Complementary information of the proteins is provided by different separation modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (cIEF), and sodium dodecyl sulphate-capillary gel electrophoresis (SDS-CGE). A powerful method for protein charge heterogeneity analysis is cIEF. In this experimental setup, the proteins in the sample solution that initially fi ll up the whole capillary, are focused into sharp bands according to their isoelectric points in a pH gradient along the capillary [1]. The pH gradient is stabilised by carrier ampholytes. They are complex mixtures of small (200 – 1,200 Da) amphoteric molecules that are good carriers of conductivity and buffering capacity at their respective isoelectric point (pI) [2]. The choice of the CA brand is an important consideration for every cIEF experiment because the composition of CAs varies [2], and infl uences the separation performance. CAs with different chemical structures and ionisable groups have been introduced and marketed under trade names such as Pharmalyte, Servalyt and AESlyte. This study shows the impact of different commercially available carrier ampholyte brands on the performance of a high resolution cIEF method for charge heterogeneity analysis of monoclonal antibody (mAb) samples (3,4,5]. The fl uorocarbon-coated capillaries used in this study proved to have a robust and reliable performance in cIEF combined with an exceptional longevity [6].


Experimental Materials


Methyl cellulose, urea, L-arginine, iminodiacetic acid and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma Aldrich (St. Louis, MO, USA). Hydrochloric acid and glacial acetic acid were sourced from Merck Millipore (Darmstadt, Germany) and phosphoric acid from JT Baker (Austin, TX, USA). Pharmalyte 3-10 were from GE Healthcare (Freiburg, Germany), Servalyt 3-10 from Serva Electrophoresis (Heidelberg, Germany), HR AESlyte 3-10 and SH AESlyte 3-10 from Advanced Electrophoresis Solutions (Cambridge, Canada) and pl markers from Sciex (Framingham, MA, USA). A rat anti-DYKDDDDK mAb (part number 200474) and all other materials and instrumentation were obtained from Agilent Technologies (Waldbronn, Germany).


Sample preparation


Prior to CE analysis, the mAb test sample was desalted using Amicon Ultra 0.5 mL centrifugal fi lter devices (Merck Millipore, Darmstadt, Germany) and a buffer containing 20 mM Tris/HCl, pH 8. The protein concentration of the desalted mABS, 3.7 mg/mL, was measured with the Qubit assay (Life Technologies, Paisley, UK). Methyl cellulose containing solutions were prepared as described [7]. Sample solutions for cIEF analysis were prepared by adding the following reagents into 0.5 mL microcentrifuge vials:


• 100 µL of 0.6% MC containing 3 M urea • 4 to 12 µL of CAs pH 3-10


• 10 µL of 500 mM L-arginine (cathodic stabiliser) • 1 µL of 200 mM iminodiacetic acid (anodic stabiliser)


• 1 µL of each pl marker • 5 µL of desalted mAb


Mixtures were vortexed for 10 s, centrifuged for about 1 minute and transferred into 100 µL CE sample vials. Sample solutions were kept in the autosampler carousel of the CE instrument at about 10°C and analysed within 24 h. UV/vis absorbance spectra of tenfold diluted CA stock solutions in water were recorded using a Nanodrop 1000 spectrophotometer (Thermo Fischer Scientifi c, Waltham, MA, USA) with a path length of 1 mm.


CE conditions


For all CE runs, an Agilent 7100 CE instruments equipped with an external water bath set to 6°C, a 280 nm high pass detector fi lter assembly and 4 bar external pressure were used. A µSIL-FC capillary with an inner diameter of 50 µm was cut at both ends at a distance of 8.5 cm and 24.5 cm from the detection window, equipped with a green alignment interface and fi tted into the capillary cassette. Once a day and after cleaning, the capillaries were conditioned as follows:


High pressure fl ush at 3.5 bar 350 mM acetic acid for 5 min Water for 2 min 0.5% MC for 5 min


Prior to every run, capillaries were conditioned as follows: High pressure fl ush at 3.5 bar 4.3 M urea solution for 3 min Water for 2 min


Samples were injected by applying 2 bar high pressure for 100 seconds, followed by a water dip of both inlet and outlet.


Focusing was done for 10-12 minutes at 25 kV with 200 mM phosphoric acid as anolyte and 300 mM NaOH as catholyte. For chemical mobilisation, the outlet vial was exchanged for 350 mM acetic acid and 30 kV was applied for 28 to 30 minutes. After each run, a high-pressure fl ush at 3.5 bar with water was done for 2 min.


After every 6 runs, the capillaries were cleaned by fl ushing them at 1 bar with 0.1 M NaOH for 2 minutes and with water for 30 minutes. Prior to storage, the capillaries were fl ushed at 1 bar with water for 20 minutes, with methanol for 5 minutes, then dried (5-minute fl ush from an empty vial). All fl ushes were done in forward direction (i.e. pressure was applied to the inlet vial). The capillary temperature was kept at 20ºC. The detection wavelength was 280/20 nm, the reference wavelength 550/100 nm and the response time 2 seconds. For all reagents 2 ml glass vials were used. The fi ll volume was 1.6 ml, except for the waste vials which were empty. All reagent vials were exchanged after six runs. Electrodes were inspected daily for the accumulation of dirt in the upper funnel, and, if necessary, cleaned as described in the user manual.


Data processing


Apparent isoelectric points were calculated by linear regression analysis of pl marker versus migrations time in MS Excel. Only pl marker 5.5 and 7.0 data for were used for the calculation. Relative peak area values (in %) were calculated with time corrected areas. Intermediate precisions were calculated with the Analyse-it for Excel statistics software package (Analyse-it Software, Leeds, UK).


INTERNATIONAL LABMATE - APRIL 2017


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  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68