Aggregation of human plasma and of human blood induced in vitro by filgrastim originator product; effect of PEGylation.

We herein report that filgrastim product Neupogen® and the filgrastim formulation buffer induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous erythrocyte aggregates when mixed with human blood, similar to the aggregation induced by pegfilgrastim and by pegfilgrastim buffer [T. Arvinte, E. Poirier, N. Ersayin, G. Darpin, A. Cudd, J. Dowd, S. Brokx, Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products, Eur. J. Pharmaceut. Biopharmaceut. (2023), doi: 10.1016/j.ejpb.2023.10.019]. The data identify the filgrastim buffer (which is practically the same in filgrastim and pegfilgrastim products) as the main driver of human plasma and blood aggregation. Kinetic experiments showed differences in the extent of plasma aggregation induced by a filgrastim product manufactured in EU and one manufactured in USA. Human donor variability in the plasma aggregation induced by filgrastim was observed. To study the effect of PEGylation of the filgrastim protein on plasma aggregation we compared filgrastim (Neupogen®) with pegfilgrastim (Neulasta®) solutions at the same protein concentration. These data show that PEGylation has a beneficial effect in inhibiting to an extent plasma aggregation. Interestingly, 20 kDa polyethylene glycol in the filgrastim buffer induced more plasma aggregation compared to the buffer, similar to the aggregation induced by pegfilgrastim. For intravenous infusion filgrastim solutions (300 µg/ml, vials only) may be diluted in 5 % dextrose from a concentration of 300 µg/ml to 5 µg/ml. Aggregation of human plasma was also induced by filgrastim solutions diluted in 5 % dextrose to 50 µg/ml, 15 µg/ml and 5 µg/ml filgrastim, as well as by the filgrastim buffer similarly diluted in 5 % dextrose (1:6, 1:20 and 1:60 dilution). These data show that filgrastim solutions used for intravenous administration in patients induce human plasma aggregation in vitro. Such aggregation phenomena may be related to known infusion side effects of filgrastim therapy.

Postsecondary Faculty Attitudes and Beliefs about Writing-Based Pedagogies in the STEM Classroom.

Writing is an important skill for communicating knowledge in science, technology, engineering, and mathematics (STEM) and an aid to developing students' communication skills, content knowledge, and disciplinary thinking. Despite the importance of writing, its incorporation into the undergraduate STEM curriculum is uneven. Research indicates that understanding faculty beliefs is important when trying to propagate evidence-based instructional practices, yet faculty beliefs about writing pedagogies are not yet broadly characterized for STEM teaching at the undergraduate level. Based on a nationwide cross-disciplinary survey at research-intensive institutions, this work aims to understand the extent to which writing is assigned in undergraduate STEM courses and the factors that influence faculty members' beliefs about, and reported use of, writing-based pedagogies. Faculty attitudes about the effectiveness of writing practices did not differ between faculty who assign and do not assign writing; rather, beliefs about the influence of social factors and contextually imposed instructional constraints informed their decisions to use or not use writing. Our findings indicate that strategies to increase the use of writing need to specifically target the factors that influence faculty decisions to assign or not assign writing. It is not faculty beliefs about effectiveness, but rather faculty beliefs about behavioral control and constraints at the departmental level that need to be targeted.

Epitope Mapping for a Preclinical Bevacizumab (Avastin) Biosimilar on an Extended Construct of Vascular Endothelial Growth Factor A Using Millisecond Hydrogen-Deuterium Exchange Mass Spectrometry.

The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.

Part 2: Physicochemical characterization of bevacizumab in 2 mg/mL antibody solutions as used in human i.v. administration: Comparison of originator with a biosimilar candidate.

The physicochemical properties of Avastin® manufactured in the USA (Originator USA) and in Europe (Originator EU) and ABX-BEV, a bevacizumab biosimilar drug product candidate produced by Apobiologix Inc., were characterized at a clinically relevant concentration of 2 mg/mL following dilution of the 25 mg/mL drug products with 0.9% NaCl. Measurements using 14 orthogonal analytical methods performed within 5 h after dilution showed good similarity of the three antibodies as regards secondary structure, conformation, aggregation properties, subvisible and visible particles. No significant protein aggregation was observed within 5 h at 24 °C in the 2 mg/mL bevacizumab solutions. The same solutions that were measured within 5 h after dilution were analyzed again after 24 h overnight refrigeration at 2-8 °C: all 2 mg/mL bevacizumab solutions showed an increase in the number and sizes of aggregates, especially the particles larger than 10 µm and 25 µm. The data show the very good similarity in the physicochemical properties of ABX-BEV with Originators USA and EU at a clinically relevant concentration.

Part 1: Physicochemical characterization of bevacizumab in undiluted 25 mg/mL drug product solutions: Comparison of originator with a biosimilar candidate.

The biosimilarity assessment of the physicochemical properties of high-concentration biopharmaceuticals is usually performed with measurements on diluted solutions, at concentrations below 1 mg/mL. In this study 13 orthogonal, spectroscopy and particle size determination methods were used to characterize the structure and aggregation of undiluted, 25 mg/mL bevacizumab drug products Avastin® manufactured in the USA and in Europe, and ABX-BEV, a bevacizumab biosimilar candidate produced by Apobiologix Inc. Secondary structure, conformation and the potential occurrence of chemical degradation of the monoclonal antibodies were characterized and compared using infrared spectroscopy, intrinsic fluorescence and ANS fluorescence spectroscopy. Protein aggregation and particulate matter in the monoclonal antibody solutions were compared using UV-Vis absorbance, 90° light-scattering, nanoparticle tracking analysis, Nile red fluorescence microscopy, particle flow imaging, ultrasound resonance technology and a new scanner-based method that visualizes protein aggregates inside unopened vials. A data wheel representation was used to plot in one figure the results from the multiple analytical methods and to highlight differences between samples. The 25 mg/mL Avastin® drug product is stored at 2-8 °C during its 2-year shelf life. After a thermal stress of 4 weeks at 40 °C the ABX-BEV solution was turbid, containing particles of 20-100 µm diameter, accompanied by strong changes in antibody structural properties. Characterization of unstressed samples stored at 2-8 °C showed that the physicochemical properties of bevacizumab in ABX-BEV and the two originator drug products were similar, the observed differences between the originators being in the same range as those between ABX-BEV and the originator. To investigate the similarity of the antibodies under stress conditions, a freeze-thaw study was performed. Although freeze-thawing of bevacizumab products is prohibited by the package insert, after two freeze-thaw cycles (24 °C to -80 °C) small changes in the structural and aggregation properties of bevacizumab were observed, changes that were similar for the originator and ABX-BEV. Our study showed a good similarity of the investigated physicochemical properties of bevacizumab in originator and ABX-BEV products. It also provides an analytical approach, based on orthogonal methods, to compare high-concentration formulations of monoclonal antibodies.

Student Learning Dispositions: Multidimensional Profiles Highlight Important Differences among Undergraduate STEM Honors Thesis Writers.

Various personal dimensions of students-particularly motivation, self-efficacy beliefs, and epistemic beliefs-can change in response to teaching, affect student learning, and be conceptualized as learning dispositions. We propose that these learning dispositions serve as learning outcomes in their own right; that patterns of interrelationships among these specific learning dispositions are likely; and that differing constellations (or learning disposition profiles) may have meaningful implications for instructional practices. In this observational study, we examine changes in these learning dispositions in the context of six courses at four institutions designed to scaffold undergraduate thesis writing and promote students' scientific reasoning in writing in science, technology, engineering, and mathematics. We explore the utility of cluster analysis for generating meaningful learning disposition profiles and building a more sophisticated understanding of students as complex, multidimensional learners. For example, while students' self-efficacy beliefs about writing and science increased across capstone writing courses on average, there was considerable variability at the level of individual students. When responses on all of the personal dimensions were analyzed jointly using cluster analysis, several distinct and meaningful learning disposition profiles emerged. We explore these profiles in this work and discuss the implications of this framework for describing developmental trajectories of students' scientific identities.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass-spectrometry-based approach.

The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass-spectrometry-based workflow that allows rapid and facile molecular characterization of antibody-based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time-resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre-clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance-derived kinetic and equilibrium binding parameters.

Understanding the Complex Relationship between Critical Thinking and Science Reasoning among Undergraduate Thesis Writers.

Developing critical-thinking and scientific reasoning skills are core learning objectives of science education, but little empirical evidence exists regarding the interrelationships between these constructs. Writing effectively fosters students' development of these constructs, and it offers a unique window into studying how they relate. In this study of undergraduate thesis writing in biology at two universities, we examine how scientific reasoning exhibited in writing (assessed using the Biology Thesis Assessment Protocol) relates to general and specific critical-thinking skills (assessed using the California Critical Thinking Skills Test), and we consider implications for instruction. We find that scientific reasoning in writing is strongly related to inference, while other aspects of science reasoning that emerge in writing (epistemological considerations, writing conventions, etc.) are not significantly related to critical-thinking skills. Science reasoning in writing is not merely a proxy for critical thinking. In linking features of students' writing to their critical-thinking skills, this study 1) provides a bridge to prior work suggesting that engagement in science writing enhances critical thinking and 2) serves as a foundational step for subsequently determining whether instruction focused explicitly on developing critical-thinking skills (particularly inference) can actually improve students' scientific reasoning in their writing.

A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim.

BACKGROUND: Recombinant human granulocyte-colony stimulating factor (G-CSF, filgrastim) is used primarily to reduce incidence and duration of severe neutropenia and its associated complications in cancer patients that have received a chemotherapy regimen. The pegylated form of filgrastim, "pegfilgrastim", is a long-acting form that requires only a once-per-cycle administration for the management of chemotherapy-induced neutropenia. Apobiologix, a division of ApoPharma USA, Inc., and Intas Pharmaceuticals Limited have co-developed a proposed pegfilgrastim biosimilar to US-licensed pegfilgrastim. METHODS: The analytical similarity of Apobiologix pegfilgrastim and US-licensed pegfilgrastim with respect to their physicochemical profile was established using a wide range of rigorous orthogonal analytical techniques. Biological function was compared using receptor binding analyses, in vitro proliferation assays, and in vivo hematopoietic progenitor mobilization. RESULTS: Apobiologix pegfilgrastim and the US-licensed pegfilgrastim reference product were found to be highly similar analytically with respect to molecular mass, primary, secondary and tertiary protein structures, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro and in vivo bioactivity. CONCLUSION: These studies provide robust evidence supporting the structural and functional similarity between Apobiologix pegfilgrastim and the US-licensed reference pegfilgrastim, and hence their biosimilarity.

Concept Maps for Improved Science Reasoning and Writing: Complexity Isn't Everything.

A pervasive notion in the literature is that complex concept maps reflect greater knowledge and/or more expert-like thinking than less complex concept maps. We show that concept maps used to structure scientific writing and clarify scientific reasoning do not adhere to this notion. In an undergraduate course for thesis writers, students use concept maps instead of traditional outlines to define the boundaries and scope of their research and to construct an argument for the significance of their research. Students generate maps at the beginning of the semester, revise after peer review, and revise once more at the end of the semester. Although some students revised their maps to make them more complex, a significant proportion of students simplified their maps. We found no correlation between increased complexity and improved scientific reasoning and writing skills, suggesting that sometimes students simplify their understanding as they develop more expert-like thinking. These results suggest that concept maps, when used as an intervention, can meet the varying needs of a diverse population of student writers.

Teaching and physics education research: bridging the gap.

Physics faculty, experts in evidence-based research, often rely on anecdotal experience to guide their teaching practices. Adoption of research-based instructional strategies is surprisingly low, despite the large body of physics education research (PER) and strong dissemination effort of PER researchers and innovators. Evidence-based PER has validated specific non-traditional teaching practices, but many faculty raise valuable concerns toward their applicability. We address these concerns and identify future studies required to overcome the gap between research and practice.

On-line Measurements and Control of Viable Cell Density in Cell Culture Manufacturing Processes using Radio-frequency Impedance.

In this work, radio-frequency (RF) impedance is reviewed as a method for monitoring and controlling cell culture manufacturing processes. It is clear from the many publications cited that RF Impedance is regarded as an accurate and reliable method for measuring the live cell bio-volume both on-line and off-line and the technology is also sutable for animal cells in suspension, attached to micro-carriers or immobilized in fixed beds. In cGMP production, RF Impedance is being used in three main areas. Firstly, it is being used as a control instrument for maintaining consistent perfusion culture allowing the bioreactor to operate under optimum conditions for maximum production of recombinant proteins. In the second application it has not replaced traditional off-line live cell counting techniques but it is being used as an additional monitoring tool to check product conformance. Finally, RF Impedance is being used to monitor the concentration of live cells immobilized on micro-carriers or packed beds in cGMP processes where traditional off-line live cell counting methods are inaccurate or impossible to perform.

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates.

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of approximately 5 x 10(6) cells mL(-1). Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell(-1) day(-1). Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized ( approximately 40 mg L(-1)) at 0.2 nL cell(-1) day(-1). The volumetric protein productivity ( approximately 60 mg L(-1) day(-1) was maximized above 0.3 nL cell(-1) day(-1). The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.