Monitoring clearance of extractables and leachables from Single-Use Technologies by NMR

Process validation at Genentech requires NMR methods capable of detecting and reliably quantifying trace levels of process impurities and leachables. Measurements must be done in solutions which are far from ideal for NMR. Depending on the application, high protein and buffer concentrations, along with the presence of water, necessitate an approach to NMR measurements and data interpretation which differs greatly from traditional NMR methods. Here, we show how we deal with these complications, and how our methods may be applied to measuring the concentrations of leachables from single-use products

Models for predicting powder-polymer properties and their use in injection molding simulations of aluminum nitride

Powder injection molding (PIM) is widely used to manufacture complex-shaped ceramic and metal components in high production volumes. In order to design and fabricate PIM components, it is important to know a number of material properties at different powder- polymer compositions. In this thesis, several predictive models for estimating rheological, thermal and mechanical properties as a function of powder-polymer mixtures were evaluated using experimental data obtained from the literature. Based on this survey, models were selected for predicting rheological, thermal and mechanical properties for aluminum nitride-polymer mixtures at various volume fractions of powder using experimental measurements of unfilled and filled polymers. The material properties were estimated for two aluminum nitride powder-polymer mixtures and used in mold-filling simulations. These results will provide new perspectives and design tools for identifying useful material compositions, component geometry attributes, and process parameters while eliminating expensive and time-consuming trial-and-error practices prevalent in PIM

Headache be gone: Clearance of extractables and leachables in single-use technologies through ultrafiltration/diafiltration

Application of single-use technologies in biopharmaceutical manufacturing can be driven by several factors such as reduced capital costs, reduced risk of cross-contamination, increased process flexibility, and reduced cleaning validation. However, implementation of single-use technologies have been restricted due to a number of concerns, with the most commonly cited being the presence of extractables and leachables (E/L) from single-use technologies. In general, overly conservative estimates of E/L are used in the risk assessment due to lack of data on clearance, resulting in a time-consuming, costly, and extensive E/L assessment for single-use technologies. A proof-of-concept study is presented here to simplify these E/L assessments for qualification and implementation of single-use technologies in biopharmaceutical manufacturing. Results from the study indicated clearance of defined E/L in protein solutions. However, unexpected clearance phenomena were observed for specific groups of E/L, which will be discussed in detail

A Radio Flare in the Long-Lived Afterglow of the Distant Short GRB 210726A: Energy Injection or a Reverse Shock from Shell Collisions?

We present the discovery of the radio afterglow of the short $\gamma$-ray burst (GRB) 210726A, localized to a galaxy at a photometric redshift of $z\sim 2.4$. While radio observations commenced $\lesssim 1~$day after the burst, no radio emission was detected until $\sim11$~days. The radio afterglow subsequently brightened by a factor of $\sim 3$ in the span of a week, followed by a rapid decay (a ``radio flare''). We find that a forward shock afterglow model cannot self-consistently describe the multi-wavelength X-ray and radio data, and underpredicts the flux of the radio flare by a factor of $\approx 5$. We find that the addition of substantial energy injection, which increases the isotropic kinetic energy of the burst by a factor of $\approx 4$, or a reverse shock from a shell collision are viable solutions to match the broad-band behavior. At $z\sim 2.4$, GRB\,210726A is among the highest redshift short GRBs discovered to date as well as the most luminous in radio and X-rays. Combining and comparing all previous radio afterglow observations of short GRBs, we find that the majority of published radio searches conclude by $\lesssim 10~$days after the burst, potentially missing these late rising, luminous radio afterglows.Comment: 28 pages, 10 figures, submitted to Ap

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse