High Molecular Weight Species Research Articles

Towards a lumped approach for solid plastic waste gasification: Polystyrene pyrolysis

In a circular economy perspective, solid plastic wastes (SPW) are a valuable source of chemicals, energy vectors and fuels. Chemical recycling through pyrolysis, gasification, and partial oxidation are promising alternatives to incineration and landfill disposal, allowing efficient exploitation of SPW. Modelling the combustion and gasification both at the particle and reactor scale requires first the definition of a suitable condensed phase pyrolysis mechanisms for each constituent. This work proposes a semi-detailed kinetic model for polystyrene (PS) pyrolysis. Following our recent work on polyethylene (PE) and polypropylene (PP), the model is based on the functional group approach firstly developed for polyvinylchloride (PVC) and biomass pyrolysis aiming at a consistent description of a wide palette of SPW components. The proposed approach retains an accurate description of low molecular weight species while representing high molecular weight species through their chemical functionalities only. The resulting liquid-phase model accounts for 34 species and 440 reactions and is attached to this paper together with the complementary gas-phase model. Model validation by a comprehensive comparison with experimental data proves its capability of predicting both mass loss and product distribution profiles with comparable accuracy to more expensive detailed models from the literature. The proposed model, together with other available subsets in the CRECK kinetic framework (i.e., biomass, PVC, PE, PP), offers a powerful tool for investigating key aspects in thermochemical recycling technologies thus supporting optimal reactor design. These aspects include mixture interactions (SPW and SPW/biomass mixtures), secondary cracking, catalytic, and gasification reactions.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.Graphical

TOI-561 b: A Low-density Ultra-short-period “Rocky” Planet around a Metal-poor Star

TOI-561 is a galactic thick-disk star hosting an ultra-short-period (0.45-day-orbit) planet with a radius of 1.37 R ⊕, making it one of the most metal-poor ([Fe/H] = −0.41) and oldest (≈10 Gyr) sites where an Earth-sized planet has been found. We present new simultaneous radial velocity (RV) measurements from Gemini-N/MAROON-X and Keck/HIRES, which we combined with literature RVs to derive a mass of M b = 2.24 ± 0.20 M ⊕. We also used two new sectors of TESS photometry to improve the radius determination, finding R b = 1.37 ± 0.04 R ⊕ and confirming that TOI-561 b is one of the lowest-density super-Earths measured to date (ρ b = 4.8 ± 0.5 g cm−3). This density is consistent with an iron-poor rocky composition reflective of the host star’s iron and rock-building element abundances; however, it is also consistent with a low-density planet with a volatile envelope. The equilibrium temperature of the planet (∼2300 K) suggests that this envelope would likely be composed of high mean molecular weight species, such as water vapor, carbon dioxide, or silicate vapor, and is likely not primordial. We also demonstrate that the composition determination is sensitive to the choice of stellar parameters and that further measurements are needed to determine whether TOI-561 b is a bare rocky planet, a rocky planet with an optically thin atmosphere, or a rare example of a nonprimordial envelope on a planet with a radius smaller than 1.5 R ⊕.

The effect of kosmotropic salts on aggregate removal from monoclonal antibodies in mixed-mode anion exchange chromatography

Efficacious and safe application of recombinant proteins in humans as drugs depends on their high purity accomplished by downstream processing. The present study evaluates the effects of kosmotropic salts used in the flow-through mode of mixed-mode anion exchange (MMAEX) chromatography to remove high molecular weight (HMW) impurities efficiently from monoclonal antibody (mAb) products. For this purpose, the impact of different kosmotropic salts at varying concentrations along with changes of the pH were systematically assessed by high throughput screens on a set of mAbs differing in their relative hydrophobicity. In contrast to the more hydrophobic mAbs, for the more hydrophilic mAbs, a clear and consistent removal of HMW species to values > 80% (achieving up to 2.5-fold improvement) occurred with the addition of increasing amounts of a kosmotropic salt (sodium chloride, sodium sulfate, ammonium sulfate, potassium chloride and potassium sulfate) to the loads applied to MMAEX chromatography. Overall, the herein described effect on the removal of HMW species by MMAEX resins was specific for kosmotropic salts, independent of conductivity, and facilitated by slightly basic pH conditions. These findings were confirmed at lab-scale operation achieving > 99% purity, indicating the potential to implement MMAEX in combination with kosmotropic salts as a polishing step for hydrophilic mAbs.

Towards a lumped approach for solid plastic waste gasification: Polyethylene and polypropylene pyrolysis

In a circular economy perspective, solid plastic wastes (SPW) can become a valuable source of chemicals, energy vectors and fuels through pyrolysis, gasification, and partial oxidation technologies, but their modelling requires first the definition of suitable condensed phase pyrolysis mechanisms for each constituent. This work proposes a semi-detailed kinetic model for polyethylene (PE) and polypropylene (PP) pyrolysis based on the functional group approach implemented for polyvinylchloride (PVC) and biomass pyrolysis to consistently address mixture modelling. This approach distinguishes polymeric chains in High Molecular Weight species, represented through their chemical functionalities, and Low Molecular Weight species, described with accuracy comparable to literature detailed models, employing the reaction classes proposed in the scientific literature. Several validated lumping techniques are introduced to reduce the model computational cost, and the resulting liquid-phase model accounts for 74 species for PE and 126 species for PP. Model validation is carried out by an extensive comparison with experimental data proving the soundness of the approach and the model capability of predicting mass-loss and product distribution profiles with similar accuracy to more expensive detailed models from the literature. The proposed condensed phase approach can be extended to other polymers and coupled with other existing subsets in the CRECK kinetic framework (e.g., biomass, PVC) paving the way for unravelling mixture interactions and secondary cracking and/or gasification reactions. The model here proposed is a powerful tool to support design and optimization of SPW thermochemical recycling technologies.

Ceramic hydroxyapatite chromatography plays a critical role in bispecific antibody purification process for impurity removal.

Significant challenges exist in downstream purification of bispecific antibodies (BsAbs) due to the complexity of BsAb architecture. A unique panel of mispaired species can result in a higher level of product-related impurities. In addition to process-related impurities such as host cell proteins (HCPs) and residual DNA (resDNA), these product-related impurities must be separated from the targeted BsAb product to achieve high purity. Therefore, development of an efficient and robust chromatography purification process is essential to ensure the safety, quality, purity and efficacy of BsAb products that consequently meet regulatory requirements for clinical trials and commercialization. We have developed a robust downstream BsAb process consisting of a mixed-mode ceramic hydroxyapatite (CHT) chromatography step, which offers unique separation capabilities tailored to BsAbs, and assessed impurity clearance. We demonstrate that the CHT chromatography column provides additional clearance of low molecular weight(LMW) and high molecular weight(HMW) species that cannot be separated by other chromatography columns such as ion exchange for a particular BsAb, resulting in ≥98% CE-SDS (non-reduced) purity. Moreover, through Polysorbate-80 (PS-80) spiking and LC-MS HCP assessments, we reveal complete clearance of potential PS-80-degrading HCP populations in the CHT eluate product pool. In summary, these results demonstrate that CHT mixed-mode chromatography plays an important role in separation of product- and process-related impurities in the BsAb downstream process.

IMMU-26. HEIGHTENED LEVELS OF CIRCULATING CELL-FREE DNA CONTRIBUTE TO PERIPHERAL IMMUNOSUPPRESSION DURING GBM PROGRESSION

Abstract Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including T cell lymphopenia. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. These high molecular weight species potently inhibits T cell proliferation in vitro despite the presence of strong T cell stimulation. Our goal was to determine the identity of the immunosuppressive species in serum of glioma-bearing mice. We performed state-of-the-art comparative high throughput mass spectrometry on fractionated and multiplexed sera isolated from control and glioma-bearing mice. This proteomics analysis demonstrated histones as a top highly abundant protein in serum of glioma-bearing mice compared to naïve controls. Cell-free DNA has been recently identified as an immune suppressive factor in circulation and can be bound to histones. We, therefore, assessed cell-free DNA levels in the serum of glioma-bearing mice. We determined that sera isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Reducing the DNA content in serum, using DNAse treatment, partially improved the defect in T cell proliferation observed when T cells are exposed to serum isolated from glioma-bearing mice. These data indicate that heighted levels of circulating DNA in serum contributes to peripheral immunosuppression in GBM. These findings provide a tractable approach to investigate the cellular source and immunosuppressive effects of cell-free DNA in murine models of GBM and to devise strategies to reverse immunosuppression with the hope of improving outcomes in patients.

Development and validation of aggregates analysis method in analytical similarity assessment of HLX04 vs Avastin®

Aggregate of therapeutic antibodies is usually considered as one of the most important critical quality attributes (CQA). The propensity of aggregates formation for bevacizumab is higher than other monoclonal antibody (mAb) drugs due to its tendency of self-association via the non-covalent interaction between the Fab arm of one bevacizumab molecule and the K445 residue on the heavy chain of another bevacizumab molecule. HLX04 has been developed as a biosimilar to bevacizumab (Avastin®) by Shanghai Henlius Biotech. To perform a head-to-head similarity evaluation with respect to aggregates or higher molecular weight species (HMWS) between HLX04 and Avastin®, we developed a robust high performance liquid chromatography (SEC-HPLC) method for aggregates analysis. Our characterization data indicated that HMWS of bevacizumab were mainly composed of dimers, and the dimer formation-dissociation equilibrium was influenced by protein concentration and storage temperature. Based on the characterization data of aggregates, we optimized the key parameters for SEC-HPLC based aggregates analysis method including mobile phase components and pH, autosampler temperature, as well as incubation conditions for sample pretreatment. The developed method was applied in HLX04 and Avastin® aggregates assessment and the similarity were confirmed among HLX04, China-sourced, and Europe-sourced Avastin® using both the pharmaceutical dosage forms and forced degradation samples. The method was also validated per ICH Q2 (R1) guidelines by challenging the parameters including specificity, accuracy, precision, linearity, range, limit of quantitation, and robustness. The validated method was applied in release test and stability study of HLX04 samples generated from commercial manufacturing process.

A macroporous resin purification process to obtain food-grade phlorotannin-rich extracts with α-glucosidase inhibitory activity from Chilean brown seaweeds: An UHPLC-MSn profiling

An efficient macroporous resin purification process was designed to obtain food-grade phlorotannin-rich extracts from the seaweeds Durvillaea incurvata and Lessonia spicata. Phlorotannins were profiled to relate structures with the α-glucosidase inhibitory activity of the extracts. Liquid chromatography-mass spectrometry was applied for tentative identification. The best phlorotannin purification performance was achieved with HP-20 resin and elution with 80% v/v ethanol. This is the first study that demonstrates the effectivity of HP-20 resin for removing potentially toxic elements (As, Cd) from seaweed extracts. Fucols/phlorethols/fucophlorethols isomers up to 4 phloroglucinol units (PGU) were the most representative phlorotannins. High molecular weight species (11–21 PGU), although in low abundances, were detected in D. incurvata for the first time. Eckols, carmalols, fuhalols, phenolic acids, and flavonoids were also detected. Some extracts’ potent α-glucosidase inhibitory activities were related to their high phlorotannin abundances, the presence of phlorotannins of a high degree of polymerization, and the phlorotannins class.

Resistance to unfolding by acidic pH and resistance to lysosomal degradation explains disease-association of HLA-B27 subtypes

Several hypotheses have been proposed to explain the high rate of disease association of HLA-B27 with ankylosing spondylitis (AS), including formation of disulfide-bonded dimers and misfolding of the heavy chain (HC), involving formation of high molecular weight (HMW) multimers. Recently, we have shown that the HMW entities of non-disease associated (non-DA) subtypes cause activation of endosomal-lysosomal pathways, while disease-associated (DA) subtypes of HLA-B27 cause activation of autophagy and unfolded protein response (UPR) pathways. In this paper, we seek an explanation for the failure of these pathways to degrade the HMW entities of DA subtypes of HLA-B27, using a combination of in vitro assays, using extracellular domains of heavy chains (EDHC), as well as in vivo assays, using stable transfectants of the full lengths of heavy chains (FLHC) of DA and non-DA subtypes. Our data shows that both DA and non-DA subtypes form HMW entities. However, non-DA HMW entities display far greater levels of degradation than DA HMW species. Non-DA EDHC display greater loss of structure at lysosomal pH in vitro. This was confirmed by experiments showing that (i) DA FLHCs co-localize with LAMP1, and (ii) induction of autophagy by rapamycin causes significant decrease in levels of non-DA HMW entities, but not that of DA HMW entities. These results point towards lack of facile lysosomal clearance of FLHCs of DA subtypes, suggesting that disease association of HLA-B27 subtypes is correlated with higher persistence of HMW entities in the low pH of lysosomes, with higher potential to trigger immune response.

Effective flow-through polishing strategies for knob-into-hole bispecific antibodies

Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4–6% and ~ 1%, respectively, to meet therapeutical requirement at 30–60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56–87%.Graphical

Host cell protein identification in monoclonal antibody high molecular weight species

High molecular weight (HMW) species are product-related variants that may impact therapeutic product safety and efficacy. Therefore, HMW species and aggregates are considered critical quality attributes and their levels should be closely monitored and controlled during drug development, commercial manufacturing, and shelf-life storage period for therapeutic monoclonal antibody drug products. Various biophysical and analytical methods have been developed to characterize the HMW species to understand their mechanisms of formation and assess potential product risk. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs are identified in the enriched HMW than in the DS, thus demonstrating a potential interaction between HCPs and HMW. Certain HCPs, including commonly detected HCPs and problematic HCPs, were enriched in HMW fractions. Especially, the most abundant HCP from mAb1, CC motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The CC motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the CC motif chemokine level in the final mAb1 DS. Our findings demonstrate that some HCPs are more preferentially bound to HMW species and this finding may provide a new opportunity for removing HCPs in downstream purification steps.

Robustness assessment of cation-exchange chromatography with in-silico peak deconvolution in monoclonal antibody purification

An in-silico tool with peak deconvolution for preparative chromatograms was developed for assessing the robustness for peak fluctuations depending on loaded sample quality. The peak positions of high-molecular-weight species (HMWS), target monoclonal antibody (Main), and low-molecular-weight species (LMWS) on a chromatogram of cation-exchange chromatography (CEX) were identified by second derivative processing. The deconvoluted peaks of HWMS, Main, and LMWS were calculated by a fitting algorithm at each identified peak position. The height of deconvolved peaks of each component was varied to mimic the change of loaded sample quality in the chromatogram, and the quality of the pooled fraction obtained from the mimic chromatogram was estimated. In addition, the relationship between the loaded sample quality and the pooled sample quality was shown to evaluate the robustness of chromatographic purification process for peak fluctuations depending on loaded sample quality. A major advantage of this robustness assessment method is to be performed using the results of only a single wet-lab experiment. We considered that it is a useful in-silico tool for rapidly assessing chromatography robustness and also product quality depending on upstream process required to achieve the target quality in the pooled fraction, at the early stage of biological pharmaceutical product development, especially monoclonal antibodies

Partial deoxygenation of pyrolysis intermediates toward fossil fuel miscible bioliquids

AbstractCatalytic fast pyrolysis of sunflower seed shells was performed using a two‐stage fixed bed reactor to produce partially deoxygenated bio‐oils. Ketonization and aldol condensation reactions were performed in a dual bed reactor using a CeO2 catalyst followed by a bifunctional Cu/Ni/ZrO2 catalyst. An ethanol‐saturated N2 stream was supplied to increase the H/C ratio of the pyrolysis intermediates. The oxygen content decreased from 23 to 13 wt % over the CeO2 fixed‐bed due to the almost complete conversion of methoxy phenols and the formation of ketones and alkyl‐substituted phenols. The Cu/Ni/ZrO2 catalyst in the presence of ethanol vapor increased the fraction of alkanes in the condensed product and favored the formation of larger ketones and higher molecular weight species. Miscibility tests showed that the partially upgraded bio‐oil could be homogeneously blended with fossil fuel oil in a 1:4 mass ratio. The oily fraction of the non‐catalytic pyrolysis bioliquid (oxygen content up to 23 wt %) was also partially miscible with the petroleum‐derived fuel.

Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSynNuc) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSynNuc in human brain cells remains controversial, yet the determination of human brain aSynNuc and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSynNuc in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSynNuc is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSynNuc was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSynNuc in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSynNuc in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype.

Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.Graphical

Abstract 179: Tumor - driven extracellular matrix remodeling influences therapeutic response

Abstract Brain cancers remain some of the most challenging tumors to treat. The goal of our research is to use a three-dimensional in vitro model to acquire in-depth understanding of the unique environment of brain tumors to find new biomarkers of disease and develop more efficient therapeutic approaches. We are interested in revealing potential associations between common genetic tumor mutations and the capacity to remodel the extracellular microenvironment. The understanding of the multiple sources of tumor heterogeneity is crucial to improve current therapeutic approaches. Glioblastomas (GBM) present a very poor prognosis, they exhibit high infiltration into brain parenchyma, impairing surgical resection and leading to recurrence. Brain extracellular matrix (ECM) plays a key role in glioblastoma invasion and therapeutic resistance. In particular, the aberrant biosynthesis of the main component of brain ECM, hyaluronic acid (HA), has been associated to pathological conditions (1). In this study, we investigate the influence of tumor extracellular HA in glioblastoma progression and resistance to a targeted tyrosine kinase inhibitor (erlotinib). We have established engineered brain tumor biomaterials based on functionalized gelatin hydrogels decorated with covalently bound HA. This biomaterial approach can monitor the response of patient-derived xenograft cell populations with different molecular signatures in combination with microfluidic devices (2, 3). We have found that the combinatorial treatment of anti-CD44 and erlotinib show synergistic effect in some EGFR overexpressing tumors containing HA (2). Moreover, we can investigate the signaling mechanisms associated to these phenotypic alterations. Different signaling pathways are altered in the tumor cells depending on the microenvironment as they respond to an EGFR and CD44 inhibitor. While the decrease in invasion and proliferation in gelatin-only matrices comes from the negative regulation of ERK pathway, HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (4). The MW and abundance of HA are mediated by the activity of HA synthases and hyaluronidases. HAS2 can produce very high molecular weight species and seems to play a significant role in cell invasion and cancer progression. In these HA-rich tumors we find a heterogeneous profile of MW distributions secreted by different tumor types, additionally we learnt that EGFR inhibition led to the secretion of high MW HA, associated to the upregulation of HAS2. We reveal here the importance of the tumor-associated HA biosynthesis as a mechanism of cancer cells to adapt to drug insults. The ability to manipulate tumor ECM can improve therapeutic outcomes and restrict glioblastoma growth and infiltration. (1) Monslow J. Front. Immunol. 2015; 6:231.(2) Pedron S. Adv. Healthcare Mater. 2017; 6:1700529.(3) Pedron S. MRS Commun. 2017; 7:442-449.(4) Pedron S. Biomaterials 2019; 219:119371. Citation Format: Edward R. Neves, Brendan A. Harley, Sara Pedron. Tumor - driven extracellular matrix remodeling influences therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 179.

Succinate Buffer in Biologics Products: Real-world Formulation Considerations, Processing Risks and Mitigation Strategies

The succinic acid/succinate system has an excellent buffering capacity at acidic pH values (4.5-6.0), promising to be a buffer of choice for biologics having slightly acidic to basic isoelectric points (pI 6 – 9). However, its prevalence in drug products is limited due to the propensity (risk) of its components to crystallize during freezing and the consequent shift in the pH which might affect the product stability. Most of these previous assessments have been performed under operational conditions that do not simulate typical drug product processing conditions. In this work, we have characterized the physicochemical behavior of succinate formulations under representative pharmaceutical conditions. Our results indicate that the pH increases by ∼ 1.2 units in 25 mM and 250 mM succinate buffers at pharmaceutically relevant freezing conditions. X-ray diffractometry studies revealed selective crystallization of monosodium succinate, which is posed as the causative mechanism. This salt crystallization was not observed in the presence of 2% w/v sucrose, suggesting that this pH shift can be mitigated by including sucrose in the formulation. Additionally, three monoclonal antibodies (mAbs) that represent different IgG subtypes and span a range of pIs (5.9 - 8.8) were formulated with succinate and sucrose and subjected to freeze-thaw, frozen storage and lyophilization. No detrimental impact on quality attributes (QA) such as high molecular weight (HMW) species, turbidity, alteration in protein concentration and sub-visible particles, was observed of any of the mAbs tested. Lastly, drug formulations lyophilized in succinate buffer with sucrose demonstrated acceptable QA profiles upon accelerated kinetic storage stability, supporting the use of succinate buffers in mAb drug products.

Modifying the Metal Surfaces in HPLC Systems and Columns to Prevent Analyte Adsorption and Other Deleterious Effects

Interactions of certain analytes with metal surfaces in high performance liquid chromatography (HPLC) instruments and columns cause a range of deleterious effects, including peak broadening and tailing, low peak areas, and the formation of new peaks due to chemical reactions. To mitigate these effects, we have developed a novel surface modification technology in which a hybrid organic/inorganic surface based on an ethylene-bridged siloxane chemistry is applied to the metal components in HPLC instruments and columns. We demonstrate the impact of this technology on peak symmetry, peak area, and injection-to-injection and column-to-column reproducibility for several metal-sensitive analytes. We also show an example of the mitigation of an on-column oxidation reaction. A variant of this technology has recently been developed for size-exclusion chromatography of proteins. An example is shown demonstrating the use of this variant applied to size-exclusion columns for the separation of a monoclonal antibody monomer and higher molecular weight species. Together, these results highlight the importance of preventing interactions of analytes with metal surfaces in HPLC in order to achieve accurate and precise results.

Heightened levels of circulating cell-free DNA predict aggressive onset of experimental glioblastoma

Abstract Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including spleen atrophy, T cell lymphopenia, and T cell sequestration within the bone marrow. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel circulating soluble factors that are found in the serum of glioma-bearing mice. This high molecular weight species potently inhibits T cell proliferation in vitro. Cell-free extracellular DNA has been recently identified as an immune suppressive factor in circulation. We, therefore, assessed cell-free DNA levels in the GL261 model. We determined that serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Serial assessment of DNA concentrations in serum of glioma-bearing mice indicated that the most prominent increase was concurrent with end-stage disease. These findings provide a tractable approach to investigate the cellular source and immune suppressive effects of cell-free DNA in the leading murine model of GBM. Supported by K99NS117799