Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®

Upton, Rosie; Migas, Lukasz; Pacholarz, Kamila J.; Beniston, Richard G.; Estdale, Siân; Firth, David; Barran, Perdita E.

doi:10.1039/c8sc05029e

Cited by 32 publications

(44 citation statements)

References 46 publications

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“…While the first one occurs at the same voltage for T0 and T1 (32.7 V), the second transition exhibits lower CIU50 values for T1 (57.1 V) than for glycosylated T0 (66.6 V) and the third transition happens at 177.8 V for T1, but only at 192.6 V for T0. As previously reported using CIU experiments [ 51 , 52 ], these results indicate that deglycosylated trastuzumab T1 is more prone to unfolding than its glycosylated counterpart, also in agreement with hydrogen-deuterium exchange (HDX) data showing increased deuterium uptake after EndoS2 deglycosylation [ 52 ]. Upon azide activation, the CIU fingerprint still looks very similar, but with slightly higher CIU50 values for the first and second transitions (37.7 and 72.7 V, respectively) ( Figure 5 C).…”

Section: Resultssupporting

confidence: 91%

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

et al. 2021

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Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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Section: Resultssupporting

confidence: 91%

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

et al. 2021

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“…No additional fragments were observed in the base peak electropherograms (Figure 9 ). The glycosylation pattern between continuous on‐demand and batchwise reconstitution showed only minor differences that are within the expected batch to batch variations for pharmaceutical mAb production (Planinc et al, 2017 ; Upton et al, 2019 ). Importantly, we did not observe significant differences in the levels of afucosylation (Figure 9 ).…”

Section: Resultsmentioning

confidence: 91%

Media on‐demand: Continuous reconstitution of a chemically defined media directly from solids

Komuczki

Dutra

Gstöttner

et al. 2021

Biotech & Bioengineering

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Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on‐demand reconstitutions directly from solids consisting of a hopper and a screw conveyor capable of feeding dry powdered media with the required precision ±5% at low dosing rates of 0.171 g min−1. A commercially available dry powdered cell culture medium was continuously fed over a duration of 12 h into a mixer which was connected to a UV‐cell for monitoring and the media were compared to a batchwise production. A comparable amino acid, carbohydrate, and osmolality profile to a batchwise reconstitution could be obtained. Cell cultivation showed comparable performance of batch and continuous reconstitution for two CHO cell lines producing the antibodies adalimumab and trastuzumab on a small and benchtop scale. In‐depth analysis of the produced antibodies showed the same glycosylation pattern, other posttranslational profiles such as methionine oxidation and deamidation compared to batchwise reconstitution. Therefore, we conclude a continuous reconstitution of the medium results in the same quality of the product. A continuous on‐demand media reconstitution will impact the supply chain and significantly reduce the floor space necessary for preparation and storage.

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“…This so-called hybrid MS strategy overcomes the shortcoming of the individual methods but generates a large amount of interdependent data, necessitating bioinformatic tools for evaluation. [19][20][21][22][23] In this study, we assess different levels of glycoprotein characterization, ranging from released glycans to intact noncovalent glycoprotein complexes. As a model for proteoform heterogeneity, we utilize human chorionic gonadotropin (hCG), a highly glycosylated protein hormone involved in early embryomaternal communication and promotion of pregnancy.…”

Section: Introductionmentioning

confidence: 99%

Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition Along Different Structural Levels of Human Chorionic Gonadotropin Employing Mass Spectrometry

Lebede¹,

Marco²,

Esser‐Skala³

et al. 2021

Preprint

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Exploration of a highly glycosylated non-covalent protein comples by mass spectreometry is a challenging task due to isobarisicy of the majority of glycoforms. Integration of data from multiple structural levels (released glycan-glycopeptide-protein subunit-protein complex) by means of computational algorithms permits unraveling the hidden diversity of the human chorionic gonadotropin heterodimer, constituting the base for the study of complex glycosylated protein assemblies.<br>

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Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®

Abstract: To quantify the measurable structural heterogeneity of a biopharmaceutical product and the effect of glycosylation on this we systematically evaluate three lots of Herceptin®, two mAb standards and an intact 5 Fc-hinge fragment.

Cited by 32 publications

References 46 publications

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Media on‐demand: Continuous reconstitution of a chemically defined media directly from solids

Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition Along Different Structural Levels of Human Chorionic Gonadotropin Employing Mass Spectrometry

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