Site-Specific Structural Changes in Long-Term-Stressed Monoclonal Antibody Revealed with DEPC Covalent-Labeling and Quantitative Mass Spectrometry.

Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum.

Effect of salt modulators on the elution behavior of insulin and the separation of product-related impurities in reversed-phase chromatography.

In this work, the effect of the salt modulators potassium chloride, ammonium chloride, ammonium sulfate, and potassium sulfate on the elution behavior of insulin in reversed-phase chromatography with ethanol as the organic modifier was investigated. Without the addition of salt modulators, insulin shows the formation of multiple peaks under non-linear loading conditions, presumably due to an aggregate formation equilibrium. Flow rate and temperature did not influence the appearance of multiple peaks.

Mechanistic modeling of cation exchange chromatography scale-up considering packing inhomogeneities.

In process development and characterization, the scale-up of chromatographic steps is a crucial part and brings a number of challenges. Usually, scale-down models are used to represent the process step, and constant column properties are assumed. The scaling is then typically based on the concept of linear scale-up.

Anti-Langmuir elution behavior of a bispecific monoclonal antibody in cation exchange chromatography: Mechanistic modeling using a pH-dependent Self-Association Steric Mass Action isotherm.

The objective of this scientific work was to model and simulate the complex anti-Langmuir elution behavior of a bispecific monoclonal antibody (bsAb) under high loading conditions on the strong cation exchange resin POROS™ XS. The bsAb exhibited anti-Langmuirian elution behavior as a consequence of self-association expressed both in uncommon retentions and peak shapes highly atypical for antibodies. The widely applied Steric Mass Action (SMA) model was unsuitable here because it can only describe Langmuirian elution behavior and is not able to describe protein-protein interactions in the form of self-association.

Two peak elution behavior of a monoclonal antibody in cation exchange chromatography as a screening tool for excipients.

Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g.

Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography.

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 1. Investigation of the influence of pH on the steric shielding factor.

In ion exchange chromatography, the Steric Mass Action (SMA) formalism is frequently used to simulate sorption processes at low and high column load conditions. To apply the SMA model for describing protein elution over wide ranges of pH, it is necessary to use pH-dependent model parameters. In the past, some publications have already described the pH-dependence of the characteristic protein charge and the equilibrium constant, while the influence of pH on the steric shielding factor has been mostly neglected.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 2. Investigation of high loading and column overloading effects.

The application of a model-based approach for industrial chromatography development requires the capability of the model to describe protein elution under high loading and overloading conditions. In a previous work, an extensive dataset was created to model the elution behavior of a bispecific antibody (bsAb) on the strong cation exchange resin POROS™ XS. Thereby, the pH-dependence of the model parameters in the Steric Mass Action (SMA) model could be examined and described over a pH range of 4.

Mechanistic modeling and simulation of a complex low and high loading elution behavior of a polypeptide in cation exchange chromatography.

The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled.

Influence of excipients in Protein A chromatography and virus inactivation.

The purification of monoclonal antibodies and Fc fusion proteins consist of several unit operations operated commonly as a platform approach, starting with Protein A chromatography. The first capture step, the following low pH virus inactivation, and subsequent ion exchange chromatography steps are mostly able to remove any impurities, like host cell proteins, aggregates, and viruses. The changes in pH and conductivity during these steps can lead to additional unwanted product species like aggregates.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8.

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state.

Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models.

Mechanistic modeling of ligand density variations on anion exchange chromatography.

Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion-exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps.

Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model.

Process development and characterization based on mathematic modeling provides several advantages and has been applied more frequently over the last few years. In this work, a Donnan equilibrium ion exchange (DIX) model is applied for modelling and simulation of ion exchange chromatography of a monoclonal antibody in linear chromatography. Four different cation exchange resin prototypes consisting of weak, strong and mixed ligands are characterized using pH and salt gradient elution experiments applying the extended DIX model.

Evaluation of differences between dual salt-pH gradient elution and mono gradient elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative-scale ion exchange chromatographic resin.

The efficiencies of mono gradient elution and dual salt-pH gradient elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno CPX. Besides giving high binding capacity, this type of opposite dual salt-pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients.

Neuroblastoma cells undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression.

Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive.

Modeling of bispecific antibody elution in mixed-mode cation-exchange chromatography.

The increasing demand for cost-efficient manufacturing of biopharmaceuticals has been the main driving force for the development of novel chromatography resins, which resulted in the development of multimodal or mixed-mode chromatographic resins. Most of them combine electrostatic and hydrophobic functionalities and are designed to deliver unique selectivity and increased binding capacities also at increased ionic strength. However, the mechanism of the protein-resin interaction in mixed-mode chromatography is still not fully understood.

Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes.

Background: Peripheral blood stem cells mobilized with granulocyte-colony-stimulating factor (G-CSF) with or without chemotherapy are routinely used for autologous hematopoietic cell transplantation. Plerixafor, a chemokine-receptor inhibitor, increases the amount of circulating CD34+ cells and improves harvest results. However, limited information is available regarding the composition of apheresis products with respect to CD34+ and lymphocyte subtypes collected after various mobilization regimens.

Genomic and transcriptional landscape of P2RY8-CRLF2-positive childhood acute lymphoblastic leukemia.

Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs.

Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype.

The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy.

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors.

Introduction: Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs.

Thermodynamic modeling of protein retention in mixed-mode chromatography: An extended model for isocratic and dual gradient elution chromatography.

An extended model is developed to describe protein retention in mixed-mode chromatography based on thermodynamic principles. Special features are the incorporation of pH dependence of the ionic interaction on a mixed-mode resin and the addition of a water term into the model which enables one to describe the total number of water molecules released at the hydrophobic interfaces upon protein-ligand binding. Examples are presented on how to determine the model parameters using isocratic elution chromatography.

Multi-color immune-phenotyping of CD34 subsets reveals unexpected differences between various stem cell sources.

Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might predict engraftment dynamics and immune reconstitution. We established a multi-color CD34 assay containing CD133, CD45RA, CD10, CD38 and CD33. We examined PBSC, donor bone marrow (BMd) and BM of patients 1 year after allografting (BM1y) regarding their CD34 subset composition, which differed significantly amongst those materials: the early CD45RA(-)CD133(+)CD38(low) subpopulations were significantly more frequent in PBSC than in BMd, and very low in BM1y.