Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles

Hernández-Jiménez, J; Martínez-Ortega, Antonio Jesús; Salmerón-García, Antonio; Cabeza, José; Prados, José Luis Paniza; Ortíz, Raúl; Navas, Natalia

doi:10.1016/j.ijbiomac.2018.06.105

Cited by 23 publications

(21 citation statements)

References 22 publications

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“…Freeze/thaw SEC experiments were carried similar to those described previously. 93 Briefly, aggregation/degradation profiles of the antibodies were tested over a 10-day period in two separate storage conditions maintained at refrigeration (4°C) or frozen at −20°C at 1 mg/mL. Samples were frozen and thawed at different days 0, 1, 2, 5, and 10 days.…”

Section: Methodsmentioning

confidence: 99%

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries

Teixeira

Erasmus

D’Angelo

et al. 2021

mAbs

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Therapeutic antibodies must have “drug-like” properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as “developability properties”: long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability. Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFβ-R2: transforming growth factor β-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda;

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Section: Methodsmentioning

confidence: 99%

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries

Teixeira

Erasmus

D’Angelo

et al. 2021

mAbs

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“…The 12 h light stress also caused ziv-AFL aggregation. It is well known that exposure to light induces aggregation in monoclonal antibodies 23,24 , and the same occurs in the ziv-AFL medicine samples, in which the dimer population clearly increased progressively over time during light exposure (from 1.9% to 12.2% (quantified by www.nature.com/scientificreports www.nature.com/scientificreports/ SE-HPLC). However, this increase in the dimer population was not detected by DLS.…”

Section: Discussionmentioning

confidence: 83%

Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation

Hermosilla

Pérez-Robles

Salmerón-García

et al. 2020

Sci Rep

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Bones 4 & natalia navas 1* Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to β-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered. Fc-fusions proteins are a well-established class of biotherapeutics. They are composed of the Fc region of the IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Although the top-selling biotherapeutics are monoclonal antibodies (mAbs), there is increasing use of therapeutic Fc-fusion proteins in medicine today 1. One of these is aflibercept (AFL), a recombinant human Fc-fusion protein that-as indicated in the assessment report of Zaltrap of the European Medicine Agency 2-"acts as a high-affinity soluble decoy receptor that preferentially binds to vascular endothelial growth factor A (VEGF-A), VEGF-B and placenta growth factor (PlGF) and prevents these factors from activating their endogenous receptors. By blocking this pathway, AFL exerts direct anti-cancer activity and boosts the anti-cancer activity of chemotherapy agents by preventing new tumour vessel growth, regressing existing tumour vessels, normalizing vasculature, negatively affecting tumour cell function, offsetting the effects of chemotherapy induction of VEGF levels and potentially inhibiting VEGF repression of dendritic cell function" 2. As a VEGF inhibitor, AFL is also classified as a VEGF-trap together with bevacizumab and ranibizumab. Cancer therapy is not the only field of medicine to benefit from AFL, which is also being used to treat several ophthalmological diseases characterized by neovascularization. As the administration to patient varies depending on the particular illness they are suffering, two different medicines are currently available, Zaltrap (Sanofi-Aventis US, LLC, Bridgewater, New Jersey, USA and Regeneron...

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“…These methods have been validated in accordance with international guidelines (validation data are presented in Supplementary data); they are commonly used in monoclonal antibody stability studies and have been proved to indicate stability. 10–14…”

Section: Methodsmentioning

confidence: 99%

Physicochemical stability study of MYL-1401O, a biosimilar of trastuzumab, following a transient temperature excursion

Guyader

Vieillard

Paul

2020

J Oncol Pharm Pract

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Introduction The extended stability of the trastuzumab biosimilar Ogivri™ (MYL-1401O; trastuzumab-dkst) was studied under different storage conditions, including following reconstitution of the lyophilized powder (21 mg/mL) but undiluted and stored in vials at 4°C; after dilution at two concentrations (0.8 and 2.4 mg/mL) in polyolefin bags and stored at 4°C; and following a three-day thermal excursion to 25°C. Methods Several methods were utilized to assess the physical and chemical stability of the drug under different storage conditions. Results At all storage conditions tested, there was no change in the tertiary structure of MYL-1401O as assessed by second-derivative ultraviolet and fluorescence-derived spectral analysis, and no evidence of oligomer formation or fragmentation was observed as assessed by gel exclusion chromatography and dynamic light scattering, confirmed by assessment of quinary structures using size-exclusion chromatography. Ion-exchange chromatography showed no significant changes in the distribution of ionic variants, particularly deamidations. Thermal denaturation curves indicated no destabilization of the three-dimensional structure after 90 days at 4°C or after thermal excursion for 72 h at 25°C. Conclusion The trastuzumab biosimilar MYL-1401O maintained its physical and chemical stability for at least 90 days at 4°C or after thermal excursion to 25°C, supporting the safe use of MYL-1401O in several real-world settings, including advanced preparation for administration or when a break in the cold cycle occurs.

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Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles

Cited by 23 publications

References 22 publications

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries

Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation

Physicochemical stability study of MYL-1401O, a biosimilar of trastuzumab, following a transient temperature excursion

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