Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing

Hosken, Brian D.; Li, Charlene; Mullappally, Berny; Co, Carl; Zhang, Boyan

doi:10.1021/acs.analchem.5b03946

Cited by 39 publications

(35 citation statements)

References 39 publications

(69 reference statements)

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“…IEF is one of the most commonly used techniques for resolving charge variants (Dada, Jaya, Valliere‐Douglass, & Salas‐Solano, ; Hazeltine et al, ; Horvath, Mun, & Laird, ; Hosken, Li, Mullappally, Co, & Zhang, ; Kaschak et al, ; Rouiller et al, ; Vanam, Schneider, & Marlow, ; Yang, Minkler, Kshirsagar, Ryll, & Huang, ; Yoon, Song, & Lee, ). IEF techniques separate molecules based on the molecules' pI and overall charge, and the detection is achieved by UV absorption at 280 nm.…”

Section: Principles Of Charge Variants Analyticsmentioning

confidence: 99%

“…This led to MS analyses that fully accounted for individual charge variant species in the basic region and partially accounted for those in the acidic region (Neill et al, 2015). Similarly, researchers have used free flow electrophoresis (FFE) to isolate specific acidic species by fractionating protein samples before analysis by IEF samples (Hosken et al, 2016;Timm, Gruber, Wasiliu, Lindhofer, & Chelius, 2010). In addition, many researchers are working to improve MS-based techniques, typically paired with IEX separation, to generate higher resolution of the specific charge variant species (Kaltashov et al, 2012).…”

Section: Areas Of Development In Analytical Techniques Include Improvedmentioning

confidence: 99%

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Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts toward developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants. Current understanding of factors affecting charge variant formation during manufacturing remains inadequate, and sometimes, even substantial commitment of resources may still not fully achieve the desired or consistent profiles. As such, this review attempts to provide a comprehensive resource for the biologics community by summarizing the impact of charge variants and CQA management, analytical methods for charge variant detection, as well as strategies in downstream and upstream PD for controlling charge variant profiles.

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Section: Principles Of Charge Variants Analyticsmentioning

confidence: 99%

Section: Areas Of Development In Analytical Techniques Include Improvedmentioning

confidence: 99%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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“…Out of 20 fractions of G. sylvestre extract obtained from Rotofor IEF, the dark-colored molecules (terpenoid saponins) were migrated and enriched at the anode end (pH 2-3) and the light-yellow clear fractions were observed at cathode end (pH 8-9) ( Figure 2). Aliquots (20 l) from each fraction (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) were separated after reducing and boiling on 15% SDS-PAGE. The Coomassie bluestained gel shows the diffused polypeptide band of about 5 kDa that is enriched in fractions 16-19 ( Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Separation of Bioactive Small Molecules, Peptide from Natural Products and Proteins from Pathogenic Microbe by Rotofor-based Preparative Isoelectric Focusing (IEF) Method

Veerapandian

Paudyal

Chang

et al. 2019

Preprint

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The primary purpose of purifying the bioactive small molecules is obtaining enough amount of specific molecules for their downstream applications. There are various methods available for serving this purpose. However, not all can afford for cost-effective purification methods, especially for complex biological samples. Here we demonstrate, the Rotofor based preparative IEF separation which shows the high possibility of separating and enriching desired molecules, including small molecules and peptides from the complex plant extract. This method is wellsuited for use at any stage of a purification scheme and has been used for complex biological samples quantification and characterization. As a proof of concept, we fractionated Gymnema sylvestre plant extract and isolated a family of terpenoid saponin molecules at the acidic end and small peptide molecule near the basic end. To our knowledge, this is a first report that Rotofor can be used to fractionate bioactive small molecules from natural product sources. We also showed effective microbial protein separation using Candida albicans fungus as a model system. The Rotofor based IEF has appealing features such as low cost, simplicity, high application potential and can be performed in any laboratory with the instrument setup.

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“…This method takes short time (∼5 min) and does not require pre‐focusing. The other is called natural pH gradient , which is accomplished by pumping a mixed ampholyte solution into the separation chamber to directly establish the pH gradient in the separation chamber. This method takes a long time (>30 min), but has a better focusing effect than the first method.…”

Section: Introductionmentioning

confidence: 99%

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

et al. 2019

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Free‐flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home‐made carrier ampholyte‐free FFIEF system was constructed via orientated migration of H+ and OH− provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R2 = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte‐free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI‐TOF‐MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris‐acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.

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Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing

Cited by 39 publications

References 39 publications

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Separation of Bioactive Small Molecules, Peptide from Natural Products and Proteins from Pathogenic Microbe by Rotofor-based Preparative Isoelectric Focusing (IEF) Method

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

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