Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody

The development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products

Subduction controls the distribution and fragmentation of Earth’s tectonic plates

International audienceThe theory of plate tectonics describes how the surface of the Earth is split into an organized jigsaw of seven large plates 1 of similar sizes and a population of smaller plates, whose areas follow a fractal distribution 2,3. The reconstruction of global tectonics during the past 200 My 4 suggests that this layout is probably a long-term feature of our planet, but the forces governing it are unknown. Previous studies 3,5,6 , primarily based on statistical properties of plate distributions, were unable to resolve how the size of plates is determined by lithosphere properties and/or underlying mantle convection. Here, we demonstrate that the plate layout of the Earth is produced by a dynamic feedback between mantle convection and the strength of the lithosphere. Using 3D spherical models of mantle convection with plate-like behaviour that match the plate size-frequency distribution observed for Earth, we show that subduction geometry drives the tectonic fragmentation that generates plates. The spacing between slabs controls the layout of large plates, and the stresses caused by the bending of trenches, break plates into smaller fragments. Our results explain why the fast evolution in small back-arc plates 7,8 reflects the dramatic changes in plate motions during times of major reorganizations. Our study opens the way to use convection simulations with plate-like behaviour to unravel how global tectonics and mantle convection are dynamically connected

Retrospective, multicenter analysis comparing conventional with oncoplastic breast conserving surgery: oncological and surgical outcomes in women with high-risk breast cancer from the OPBC-01/iTOP2 study

Introduction: Recent data suggest that margins ≥2 mm after breast-conserving surgery may improve local control in invasive breast cancer (BC). By allowing large resection volumes, oncoplastic breast-conserving surgery (OBCII; Clough level II/Tübingen 5-6) may achieve better local control than conventional breast conserving surgery (BCS; Tübingen 1-2) or oncoplastic breast conservation with low resection volumes (OBCI; Clough level I/Tübingen 3-4). Methods: Data from consecutive high-risk BC patients treated in 15 centers from the Oncoplastic Breast Consortium (OPBC) network, between January 2010 and December 2013, were retrospectively reviewed. Results: A total of 3,177 women were included, 30% of whom were treated with OBC (OBCI n = 663; OBCII n = 297). The BCS/OBCI group had significantly smaller tumors and smaller resection margins compared with OBCII (pT1: 50% vs. 37%, p = 0.002; proportion with margin <1 mm: 17% vs. 6%, p < 0.001). There were significantly more re-excisions due to R1 (“ink on tumor”) in the BCS/OBCI compared with the OBCII group (11% vs. 7%, p = 0.049). Univariate and multivariable regression analysis adjusted for tumor biology, tumor size, radiotherapy, and systemic treatment demonstrated no differences in local, regional, or distant recurrence-free or overall survival between the two groups. Conclusions: Large resection volumes in oncoplastic surgery increases the distance from cancer cells to the margin of the specimen and reduces reexcision rates significantly. With OBCII larger tumors are resected with similar local, regional and distant recurrence-free as well as overall survival rates as BCS/OBCI

Osteoporotic bone fractures: intramedullary augmentation and hybrid osteosynthesis

The increasing number of people who are living longer and have a far more active lifestyle is inevitably associated with greater numbers of fractures. Stabilization of these fractures in older patients with plates and screws is complicated by fragile bone substance, especially in osteoporotic bone, since osteosynthesis with a conventional plate depends exclusively on the holding power of the screws. Therefore, treatment requires new stabilization technologies designed for these specific tasks. A small diameter polyethylene terephthalate (PET, Dacron (R)) balloon is delivered in a minimally invasive fashion and placed within the canal, transversing the fracture. Once positioned, the balloon is expanded with a liquid monomer to fill the medullary canal. The liquid monomer is then rapidly cured using visible blue light, forming a patient-customized intramedullary implant that stabilizes the entire length of the bone in contact with the implant. The described intramedullary implant can be easily drilled in any position or location, providing a substantial increase in screw holding power. Thus, a major advantage of the technique is the possibility to augment the newly formed balloon nail with a conventional plate and screws at the primary stabilization or at any later time

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics

Effective management of bone fractures with the IlluminOss® photodynamic bone stabilization system: initial clinical experience from the European Union registry

The IlluminOss® system (IS) uses a light-curable polymer contained within an inflatable balloon catheter, forming a patient customized intramedullary implant. A registry was established in Germany and The Netherlands to prospectively collect technical and clinical outcomes in patients treated with IS for fractures of the phalange, metacarpal, radius, ulna, distal radius, fibula, clavicle and/or olecranon. Humeral, femoral, tibial and pelvic fractures were included under compassionate use. Procedural success included successful placement of the device at the target fracture site and achievement of fracture stabilization. Clinical and radiographic assessments were made postoperatively through 12 months. One hundred thirty two patients (149 fractures) were enrolled with most fractures (85%) resulting from low-energy trauma. Simple fractures predominated (47%) followed by complex (23%) and wedge (16%) fractures. Procedural success was achieved in all patients and no implants required removal or revision. Normal range of motion was realized in 87% of fractures. Radiographically, there was substantial cortical bridging, total dissolution of the fracture line, and complete fracture healing. Across a variety of fracture types, the IS provides a safe and effective approach for rapid healing and functional recovery

Sedimentological and carbonate isotope signatures to identify fluvial processes and catchment changes in a supposed impact ejecta‐dammed lake (Miocene, Germany)

The identification and distinction of fluvial from lacustrine deposits and the recognition of catchment changes are crucial for the reconstruction of climate changes in terrestrial environments. The investigated drill core succession shows a general evolution from red–brown claystones to white–grey marlstones and microcrystalline limestones, which all have previously been considered as relict deposits of an impact ejecta‐dammed lake, falling within the mid‐Miocene Climate Transition. However, recent mammal biostratigraphic dating suggests a likely pre‐impact age. Indeed, no pebbles from impact ejecta have been detected; only local clasts of Mesozoic formations, in addition to rare Palaeozoic lydites from outside of the study area. Lithofacies analysis demonstrates only the absence of lacustrine criteria, except for one charophyte‐bearing mudstone. Instead, the succession is characterized by less diagnostic floodplain fines with palaeosols, palustrine limestones with root voids and intercalated thin sandstone beds. Carbonate isotope signatures of the mottled marlstones, palustrine limestones and mud‐supported conglomerates substantiate the interpretation of a fluvial setting. Low, invariant δ18Ocarb reflects a short water residence time and highly variable δ13Ccarb indicates a variable degree of pedogenesis. Carbonate 87Sr/86Sr ratios of the entire succession show a unidirectional trend from 0.7103 to 0.7112, indicating a change of the solute provenance from Triassic to Jurassic rocks, identical to the provenance trend from extraclasts. The increase in carbonate along the succession is therefore independent from climate changes but reflects a base‐level rise from the level of the siliciclastic Upper Triassic to the carbonate‐bearing Lower to Middle Jurassic bedrocks. This study demonstrates that, when information on sedimentary architecture is limited, a combination of facies criteria (i.e. presence or absence of specific sedimentary structures and diagnostic organisms), component provenance, and stable and radiogenic isotopes is required to unequivocally distinguish between lacustrine and fluvial sediments, and to disentangle regional geological effects in the catchment and climate influences.China Scholarship Council http://dx.doi.org/10.13039/501100003398Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/50110000165

Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody

ABSTRACTThe development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics