Capillary size exclusion chromatography with picogram sensitivity for analysis of monoclonal antibodies purified from harvested cell culture fluid

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DAR), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

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“…Despite this limitation, 300 mm x 300 m i.d. SEC capillary columns were however applied for the separation of mAbs fragments [93,94].…”

Section: Secmentioning

confidence: 99%

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Bobály

Fleury-Souverain²,

Beck

et al. 2018

Journal of Pharmaceutical and Biomedical Analysis

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“…Rea et al reported the use of 300 μm id capillary SEC columns for mAb analysis purified from harvested cell culture fluid. After optimizing the fluidics to minimize system dispersion, picogram sensitivity was achieved in combination with UV detection [54]. Smoluch et al applied a 300 μm id column format for the online SEC-ESI-MS analysis of peptides in a mass range of 0.1-7 kDa [55].…”

Section: Aqueous Size-exclusion Chromatography For the Analysis Of Prmentioning

confidence: 99%

“…reported the use of 300 μm id capillary SEC columns for mAb analysis purified from harvested cell culture fluid. After optimizing the fluidics to minimize system dispersion, picogram sensitivity was achieved in combination with UV detection . Smoluch et al.…”

Section: Native Lc Modesmentioning

confidence: 99%

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

Tassi

Vos

Chatterjee

et al. 2017

J of Separation Science

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The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state‐of‐the‐art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid‐chromatographic modes that are discussed include aqueous size‐exclusion chromatography, hydrophobic interaction chromatography, and ion‐exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid‐chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody–drug conjugates is discussed.

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“…Furthermore, host cell proteins (HCPs) and cell culture medium components may complicate aggregate detection. Nowadays, investigation of protein aggregation during cell cultivation is usually performed after a Protein A capture step [ 11 - 14 ]. Protein A affinity chromatography is a powerful tool for antibody purification, but it also exposes antibodies to pH-shifts [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Direct analysis of mAb aggregates in mammalian cell culture supernatant

2014

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BackgroundProtein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). Current methods to determine aggregate formation during cell culture include size exclusion chromatography (SEC) with a previous affinity chromatography step in order to remove disturbing cell culture components. The pre-purification step itself can already influence protein aggregation and therefore does not necessarily reflect the real aggregate content present in cell culture. To analyze mAb aggregate formation directly in the supernatant of Chinese hamster ovary (CHO) cell culture, we established a protocol, which allows aggregate quantification using SEC, without a falsifying pre-purification step.ResultsThe use of a 3 μm silica SEC column or a SEC column tailored for mAb aggregate analysis allows the separation of mAb monomer and aggregates from disturbing cell culture components, which enables aggregate determination directly in the supernatant. Antibody aggregate analysis of a mAb-producing CHO DG44 cell line demonstrated the feasibility of the method. Astonishingly, the supernatant of the CHO cells consisted of over 75% mAb dimer and larger oligomers, representing a substantially higher aggregate content than reported in literature so far.ConclusionThis study highlights that aggregate quantification directly in the cell culture supernatant using appropriate SEC columns with suitable mAb aggregate standards is feasible without falsification by previous affinity chromatography. Moreover, our results indicate that aggregate formation should be addressed directly in the cell culture and is not only a problem in DSP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.

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Capillary size exclusion chromatography with picogram sensitivity for analysis of monoclonal antibodies purified from harvested cell culture fluid

Cited by 19 publications

References 13 publications

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

Direct analysis of mAb aggregates in mammalian cell culture supernatant

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