16 February / March 2019
High-Throughput Precision with On-Board Mixing for Imaged Capillary Isoelectric Focusing (iCIEF)
by Katelyn Cousteils, Jennifer Hu, Victor Li, Tiemin Huang Advanced Electrophoresis Solutions Ltd, 1600 Industrial Road, Cambridge, Ontario, N3H 4W5, Canada. Email:
tiemin@aeslifesciences.com
Imaged capillary isoelectric focusing (iCIEF) is a powerful technique used in many biopharmaceutical laboratories responsible for evaluating the charge heterogeneity of proteins. Bottlenecks occur in the manual preparation of different samples and variability of test results is introduced by the technician. Some proteins are not stable in the presence of ampholytes which must be added to the sample immediately prior to injection. In this article we have demonstrated that automated mixing of the sample by the autosampler, or on-board mixing, prior to injection delivers comparable results to manual mixing, as determined by peak area percentage and pI precision, while saving the user time spent in sample preparation.
Abbreviations
ADC, antibody-drug conjugate; CIEF, capillary isoelectric focusing; CQA, critical quality attribute; iCIEF, imaged capillary isoelectric focusing; IEF, isoelectric focusing; mAb, monoclonal antibody; PAP, peak area percentage; pI, isoelectric point; PTM, post-translational modification; RSD, relative standard deviation; WCID, whole-column image detection; UV, ultraviolet
Introduction
Monoclonal antibodies (mAbs) are an increasingly important subset of therapeutic proteins [1]. The targeted nature of mAbs and associated medicines such as antibody-drug conjugates (ADCs) offers more disease-specific biodistribution in the body when compared to traditional small molecule medicines [2]. Further, mAbs are manufactured relatively quickly and easily using hybridoma technology [3].
mAbs are produced in bioreactors but structural variation like hydrolysis, deamidation, oxidation, glycosylation, and deglycosylation will occur during production as well as degradation during storage [4]. This generates variability in the surface charge of the therapeutic proteins produced, or charge heterogeneity [5]. Charge heterogeneity is a structural critical quality attribute (CQA) of mAbs [6].
The platform technology for determining Figure 1: CEInfinite Analytical iCIEF Instrument with On-Board Mixing
and quantifying charge heterogeneity is capillary isoelectric focusing (CIEF). CIEF is a CE method in which amphoteric compounds called ampholytes generate a pH gradient when a voltage is applied. During focusing, proteins that vary in their overall charge will migrate along the gradient to where the pH is equal to their isoelectric point (pI) [7]. In CIEF there is a single point of detection where they are visualised using ultraviolet (UV) light that requires the focused proteins to be slowly pushed past the point of detection. However, the separation resolution suffers during the mobilisation step [8].
Imaged CIEF (iCIEF) uses a whole-column image detection (WCID) cartridge; the entire separation capillary is illuminated with UV
light eliminating the need to mobilise the focused protein and thus conserving the obtained resolution in the focusing step [9].
While iCIEF runs are fast, duplicates add up and reactions/degradations can occur prior to sample injection and focusing. The CEInfinite Analytical iCIEF instrument comes with a 96-well plate option for the autosampler that is capable of automated, or on-board, mixing. With on-board mixing, sensitive protein samples and isoelectric focusing (IEF) solutions remain separate in the temperature-controlled autosampler until they are mixed immediately prior to injection into the separation capillary. Less stable proteins are kept well preserved until focusing. Additionally, charge heterogeneity data such as pI or peak area percentage
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