Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

The new frontier in CHO cell line development: From random to targeted transgene integration technologies.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

Tailoring polishing steps for effective removal of polysorbate-degrading host cell proteins in antibody purification.

Ensuring the quality and safety of biopharmaceutical products requires the effective separation of monoclonal antibodies (mAbs) from host cell proteins (HCPs). A major challenge in this field is the enzymatic hydrolysis of polysorbates (PS) in drug products. This study addresses this issue by investigating the removal of polysorbate-degrading HCPs during the polishing steps of downstream purification, an area where knowledge about individual HCP behavior is still limited. We investigated the separation of different mAb formats from four individual polysorbate degrading hydrolases (CES1F, CES2C, LPLA2, and PAF-AH) using cation exchange (CEX) and mixed-mode chromatography (MMC) polishing steps. Our research identified a key challenge: The similar elution behavior of mAbs and HCPs during chromatographic separation. To investigate this phenomenon, we performed high-throughput binding screenings for recombinant polysorbate degrading hydrolases and representative mAb candidates on CEX and MMC chromatography resins. We then employed a three-step strategy that also served as a scale-up process, optimizing separation conditions and leading to the successful removal of specific HCPs while maintaining high mAb recovery rates (>96%). This strategy involved the use of surface response models and miniature columns for screening, followed by validation on larger columns using a chromatography system. Our results highlight the critical role of the inherent properties of mAbs for successful separation from HCPs. These results underscore the need to tailor the purification process to leverage the slight differences in binding behavior and elution profiles between mAbs and specific HCPs. This approach lays the foundation for developing more effective strategies for overcoming the challenge of enzymatic polysorbate degradation, paving the way for improved quality and safety in biopharmaceutical products.

Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells.

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.

When will we have a clone? An industry perspective on the typical CLD timeline.

Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre-transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line.

Multi-lipase gene knockdown in Chinese hamster ovary cells using artificial microRNAs to reduce host cell protein mediated polysorbate degradation.

A large number of companies observe polysorbate (PS) degradation and associated (sub-)visible particle formation in biological drug formulations, which compromise the stability of the drug product, ultimately posing a risk toward delivering innovative medicines to patients. The main culprits of PS degradation are hydrolytic host cell proteins (HCPs) originating from the production cell lines, which are mostly Chinese hamster ovary (CHO) cell derived. Here, a small portion of particularly difficult-to-remove HCPs-mainly lipases-cause hydrolytic cleavage of PS resulting in the accumulation of free fatty acid aggregates/particles. One possible mitigation strategy is the removal of such critical HCPs in the production cell line. Multigene regulation can be achieved via microRNAs (miRNAs) thereby serving as a smart tool to reduce the expression of different target genes using a single miRNA. To enable a tailored gene regulation of multiple specific target lipases self-designed and non-naturally occurring artificial miRNAs (amiRNA) can be designed. Based on micro-conserved regions in the mRNA sequence of two sets of target HCPs, we provide a proof-of-concept for a simultaneous multi-lipase knockdown in CHO cells using single amiRNAs. By this, we were not only able to reduce PS degradation but laid the foundation to expand this tool to other areas of cell line phenotype engineering.

In pursuit of a minimal CHO genome: Establishment of large-scale genome deletions.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories.

A Mild Method for the Activation of Cation Exchange Membranes Used in Tubular PEM Electrolyzers.

Co-extrusion of both half-cells in tubular PEM water electrolyzers can lower the costs for hydrogen production, since the number of components is reduced and the production process is simplified. However, after co-extrusion of the inner half-cell and the ion exchange membrane, the membrane is in its fluoride sulfonyl form and must be hydrolyzed to achieve the proton conductive sulfonic acid to be ready for use. Common practice is the hydrolysis using concentrated alkaline solutions, which causes a corrosion of the laminated anode electrode. We developed a less corrosive method using triethylsilanol as reactant. Tubular membranes hydrolyzed with this new procedure were characterized and tested in an electrolyzer laboratory test setup.

Droplet digital PCR: A comprehensive tool for genetic analysis and prediction of bispecific antibody assembly during cell line development.

Molecular biological methods have emerged as inevitable tools to accompany the process of cell line development for the generation of stable and highly productive manufacturing cell lines in the biopharmaceutical industry. PCR-based methods are especially useful for screening and characterization of cell lines due to their low cost, scalability, precision and propensity for multidimensional read-outs. In this study, the diverse applications of droplet digital PCR (ddPCR) as a molecular biological tool for cell line development are demonstrated. Specifically, it is shown that ddPCR can be used to enable precise, sensitive and reproducible absolute quantification of genomically integrated transgene copies during cell line development and cell bank characterization. Additionally, an amplitude multiplexing approach is applied to simultaneously run multiple assays on different genetic targets in a single reaction and advance clonal screening by measuring gene expression profiles to predict the assembly and homogeneity of difficult-to-express (DTE) proteins. The implementation of ddPCR-based assays during cell line development allows for early screening at a transcriptional level, particularly for complex, multidomain proteins, where balanced polypeptide chain ratios are of primary importance. Moreover, it is demonstrated that ddPCR-based genomic characterization improves the robustness, efficiency and comparability of absolute transgene copy number quantification, an essential genetic parameter that must be demonstrated to regulatory authorities during clinical trial and market authorization application submissions to support genetic stability and consistency of the selected cell substrate.

Reflection Machines: Supporting Effective Human Oversight Over Medical Decision Support Systems.

Human decisions are increasingly supported by decision support systems (DSS). Humans are required to remain "on the loop," by monitoring and approving/rejecting machine recommendations. However, use of DSS can lead to overreliance on machines, reducing human oversight. This paper proposes "reflection machines" (RM) to increase meaningful human control. An RM provides a medical expert not with suggestions for a decision, but with questions that stimulate reflection about decisions. It can refer to data points or suggest counterarguments that are less compatible with the planned decision. RMs think against the proposed decision in order to increase human resistance against automation complacency. Building on preliminary research, this paper will (1) make a case for deriving a set of design requirements for RMs from EU regulations, (2) suggest a way how RMs could support decision-making, (3) describe the possibility of how a prototype of an RM could apply to the medical domain of chronic low back pain, and (4) highlight the importance of exploring an RM's functionality and the experiences of users working with it.

Low-energy electron microscopy intensity-voltage data - Factorization, sparse sampling and classification.

Low-energy electron microscopy (LEEM) taken as intensity-voltage (I-V) curves provides hyperspectral images of surfaces, which can be used to identify the surface type, but are difficult to analyse. Here, we demonstrate the use of an algorithm for factorizing the data into spectra and concentrations of characteristic components (FSC3 ) for identifying distinct physical surface phases. Importantly, FSC3 is an unsupervised and fast algorithm. As example data we use experiments on the growth of praseodymium oxide or ruthenium oxide on ruthenium single crystal substrates, both featuring a complex distribution of coexisting surface components, varying in both chemical composition and crystallographic structure. With the factorization result a sparse sampling method is demonstrated, reducing the measurement time by 1-2 orders of magnitude, relevant for dynamic surface studies. The FSC3 concentrations are providing the features for a support vector machine-based supervised classification of the surface types. Here, specific surface regions which have been identified structurally, via their diffraction pattern, as well as chemically by complementary spectro-microscopic techniques, are used as training sets. A reliable classification is demonstrated on both example LEEM I-V data sets.

Optimisation of individual cardiovascular risk assessment in a German coronary artery disease cohort using a commercial test for genetic polymorphisms - a pilot study.

BACKGROUND AND AIMS: Assessment of cardiovascular risk using established risk scores such as ESC SCORE2 or PROCAM insufficiently emphasise the role of genetic factors. We hypothesise that commercially available genetic assays may provide additional information on hereditary cardiovascular risk in a timely and cost-efficient manner. METHODS: In a cohort of 51 patients treated for coronary artery disease (CAD) at University Hospital Heidelberg, Germany, a subgroup of patients with "unstable" CAD (i.e. recurrent acute coronary syndrome) was identified and compared to patients with "stable" disease (i.e. chronic coronary syndrome). Gene array analysis using a commercial assay for 15 potentially pathogenic polymorphisms revealed our cohort's genetic risk profile regarding atherosclerotic/thromboembolic events. Improvement of cardiovascular risk assessment based on established risk scores was analysed using net reclassification, logistic regression and receiver operating characteristic (ROC) analysis. RESULTS: Discriminatory capacity of traditional risk scores such as SCORE2 or PROCAM with regard to stable and unstable CAD groups was poor (ROC AUC <0.5). Patients with "unstable" CAD exhibited a significantly increased frequency of pathogenic eNOS 894 T and MTHFR 1298 C polymorphisms compared to "stable" CAD patients, and information on these polymorphisms individually as well as combinations with additional polymorphisms included in the assay such as ACE D/D or PAI-1 5 G variants markedly improved risk prediction compared to SCORE2/PROCAM alone (ROC AUC ≥0.75). CONCLUSION: Commercially available assays for genetic polymorphisms may provide valuable information on individual genetic cardiovascular risk, potentially guiding future primary and/or secondary preventative therapies for coronary artery disease.

Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell-Derived Cells.

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115.

High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells.

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.

Towards maximum acceleration of monoclonal antibody development: Leveraging transposase-mediated cell line generation to enable GMP manufacturing within 3 months using a stable pool.

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months.

Decline in Atlantic wolffish Anarhichas lupus in the North Sea: Impacts of fishing pressure and climate change.

Anarhichas lupus is a boreo-Arctic species with biological characteristics often associated with vulnerability to overexploitation. Although not commercially targeted in the North Sea, A. lupus is a bycatch species in mixed demersal fisheries. Here we provide an overview of the status of A. lupus in the North Sea, as observed from commercial landings and fishery-independent trawl survey data. A. lupus was once common across much of the central and northern North Sea but, since the 1980s, have declined in abundance, demographic characteristics (reduced size) and geographical range, with the shallower and more southerly parts of its range most impacted. A. lupus is still relatively frequent in the northern North Sea, where fishing intensity, though decreasing, is high. Bycatch through fishing remains a potential threat and, considering the likely impacts of predicted climate change on cold-water species, risks of further regional depletion and/or range contraction remain. Whether or not A. lupus is able to re-establish viable populations in former habitat in UK coastal waters is unknown. Given the lack of data, the precautionary principle would suggest that manageable pressures be minimized where the species and its habitat are at risk of further impacts, and more regular assessments of population status be undertaken.

A blueprint from nature: miRNome comparison of plasma cells and CHO cells to optimize therapeutic antibody production.

Chinese Hamster Ovary (CHO) cells are the most frequently used biopharmaceutical production hosts, although industry is presently suffering from their variable recombinant product quality, insufficient long-term stability and low productivity. Here, we present an effort to address overall cell line engineering by a novel bottom-up microRNA (miRNA) screening approach. miRNAs are small non-coding RNAs known to regulate global gene expression at the post-transcriptional level and have proved to serve as promising tools for cell line engineering for over a decade. Here the miRNome of plasma cells (PCs) has been analyzed as the natural blueprint for optimized production and secretion of antibodies. Performing comparative miRNome cross-species expression analysis of four murine/human PC-derived (PCD) and two CHO cell lines showed 147 conserved miRNAs to be differentially expressed between PCDs and CHOs. Conducting a targeted miRNA screen of this PC-specific miRNA subset revealed 14 miRNAs to improve bioprocess relevant parameters in CHO cells, among them the PC-characteristic miR-183 cluster. Finally, miRNA target prediction tools and transcriptome analysis were combined to elucidate differentially regulated lysine degradation and fatty acid metabolism pathways in monoclonal antibody (mAb) expressing CHO-DG44 and CHO-K1 cells, respectively. Thus, substantial new insights into molecular and cellular mechanisms of biopharmaceutical production cell lines can be gained by targeted bottom-up miRNA screenings.

Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

Impact of a Cold Environment on the Performance of Professional Cyclists: A Pilot Study.

The practice of physical activity in a variable climate during the same competition is becoming more and more common due to climate change and increasingly frequent climate disturbances. The main aim of this pilot study was to understand the impact of cold ambient temperature on performance factors during a professional cycling race. Six professional athletes (age = 27 ± 2.7 years; height = 180.86 ± 5.81 cm; weight = 74.09 ± 9.11 kg; % fat mass = 8.01 ± 2.47%; maximum aerobic power (MAP) = 473 ± 26.28 W, undertook ~20 h training each week at the time of the study) participated in the Tour de la Provence under cold environmental conditions (the ambient temperature was 15.6 ± 1.4 °C with a relative humidity of 41 ± 8.5% and the normalized ambient temperature (Tawc) was 7.77 ± 2.04 °C). Body core temperature (Tco) was measured with an ingestible capsule. Heart rate (HR), power, speed, cadence and the elevation gradient were read from the cyclists' onboard performance monitors. The interaction (multivariate analysis of variance) of the Tawc and the elevation gradient has a significant impact (F(1.5) = 32.2; p < 0.001) on the variables (cadence, power, velocity, core temperature, heart rate) and on each individual. Thus, this pilot study shows that in cold environmental conditions, the athlete's performance was limited by weather parameters (ambient temperature associated with air velocity) and race characteristics. The interaction of Tawc and elevation gradient significantly influences thermal (Tco), physiological (HR) and performance (power, speed and cadence) factors. Therefore, it is advisable to develop warm-up, hydration and clothing strategies for competitive cycling under cold ambient conditions and to acclimatize to the cold by training in the same conditions to those that may be encountered in competition.

The morphology of VO2/TiO2(001): terraces, facets, and cracks.

Vanadium dioxide (VO2) features a pronounced, thermally-driven metal-to-insulator transition at 340 K. Employing epitaxial stress on rutile [Formula: see text] substrates, the transition can be tuned to occur close to room temperature. Striving for applications in oxide-electronic devices, the lateral homogeneity of such samples must be considered as an important prerequisite for efforts towards miniaturization. Moreover, the preparation of smooth surfaces is crucial for vertically stacked devices and, hence, the design of functional interfaces. Here, the surface morphology of [Formula: see text] films was analyzed by low-energy electron microscopy and diffraction as well as scanning probe microscopy. The formation of large terraces could be achieved under temperature-induced annealing, but also the occurrence of facets was observed and characterized. Further, we report on quasi-periodic arrangements of crack defects which evolve due to thermal stress under cooling. While these might impair some applicational endeavours, they may also present crystallographically well-oriented nano-templates of bulk-like properties for advanced approaches.