ProteinSimple (United States)

A significant limitation of imaged capillary electric focusing (icIEF) is the inability to identify and characterize specific species in the electropherogram. This has led to the development of complementary ion-exchange chromatography (IEX)-based methods that are amenable to either fraction collection and subsequent characterization or online IEX coupled to mass spectrometry. To overcome this limitation while maintaining the use of icIEF, novel approaches, including an icIEF separation and fractionation technology (MauriceFlex, ProteinSimple), have been developed. This approach enables the fractionation of various icIEF peaks, which can then be characterized by mass spectrometry to confirm the identity of the separated charged species. Herein, the MauriceFlex technology was applied to adeno-associated viral (AAV) gene therapy products, which contain a DNA transgene packaged into a protein capsid and have shown tremendous therapeutic potential in recent years. Utilizing the MauriceFlex system, we developed an approach for the separation of charged species from AAV capsid viral proteins (VP) by icIEF and subsequent characterization by liquid chromatography and mass spectrometry (LC-MS). When applying the same sample preparation, charge profiles of AAV capsid proteins on the MauriceFlex instrument were demonstrated to be consistent with those from the original Maurice platform, the industrial gold standard. Optimization of the VP icIEF fractionation method required the development of a method for low concentration samples, optimization of mobilization conditions, enhancement of fraction recovery, and maintenance of protein stability post fractionation. Herein, we were able to successfully collect charge-separated VP fraction samples and subsequently analyze them by MS analysis. In addition, a workflow for AAV capsid protein characterization based on icIEF separation and fractionation coupled with downstream LC-MS has been established for the confirmation of VP identity and additional characterization of capsid protein heterogeneity.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Nowadays, recommendation systems are a central component in a wide range of online services. One major challenge for them is to generate suitable recommendations for individual users who differ, among other things, in their interests and preferences. In this paper, we propose a deep learning-based product-to-product recommendation system that uses product embeddings to incorporate content-based product information and user data in form of historical checkouts. On top of that, we add a filter that rearranges the models' recommendations based on the individual user preferences to a product category that is derived from past activities. In order to evaluate and show the impact of our approach, we conduct tests on historical data and an A/B-test in a real online service that offers thousands of different customer packaged goods (CPG) to millions of users. The results show that our approach delivers suitable product recommendations to users and outperforms the current system in use.

Abstract The PI3K-Akt signaling pathway modulates cell growth, survival and apoptosis, and this pathway is frequently altered in human cancers, contributing resistance to radiation and chemotherapy treatment. Akt is a target for specific inhibition and recently, a number of small molecules have been developed to improve the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2, and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of the Multiple Detection Approach to characterize the PI3K-Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immunoassays or immunoassay with total protein on the Simple Western platform using chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight via capillary electrophoresis, followed by immobilization via UV-crosslinking. Next, PI3K-Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies, to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe were removed after the detection step with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading, and/or other systematic errors. These results present the utility of the Multiple Detection Approach to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies. Citation Format: Irina G. Kazakova, Crystal Tran, Anita Silver, Jessica Dermody. Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6293.

e17565 Background: We previously reported the significant prognostic and predictive utility of pretreatment serum PD-L1 in the CCTG MA.31 breast cancer serum bank (SABCS 2018, abstr PD3-10). IL-8 (CXCL8) is a pro-inflammatory cytokine that binds to CXCR1 and CXCR2 and promotes tumor immune escape and progression. High serum IL-8 levels are associated with poor prognosis in many cancers, and have recently been reported to predict for reduced overall survival (OS) to nivolumab in lung cancer and melanoma (ASCO 2018, abstr #3025). Here we correlated plasma IL-8 and PD-L1 levels with OS in mCRPC patients. Methods: 201 metastatic CRPC patients had EDTA plasma available for this retrospective analysis. Patient eligibility included chemotherapy-naive mCRPC patients. The ELLA immunoassay platform (ProteinSimple, San Jose, CA) was utilized to quantitate plasma IL-8 and PD-L1. Cox regression assessed hazard ratio (HR) for OS using both categorical (median) and continuous (log-transformed) biomarkers. Results: In univariate analysis, higher plasma IL-8 levels were significantly associated with reduced OS when analyzed as a continuous variable (HR = 1.53; p = 0.003) and were of borderline significance at the median cutpoint (HR = 1.32; p = 0.069; 20.9 vs 31.5 mos median OS). Plasma PD-L1 levels were not significantly associated with OS when analyzed as a continuous variable (p = 0.17), but increased levels were significant when analyzed at the median cutpoint (HR = 1.36; p = 0.044; 21.9 vs 29.0 mos median OS). When plasma IL-8 and PD-L1 levels were combined (median cutpoints), plasma IL-8 high / PD-L1 high patients (n = 58) had a significantly shorter OS vs the plasma IL-8 low / PD-L1 low patients (n = 58) (HR = 1.69; p = 0.009; 19.3 vs 32.9 mos median OS, respectively). In multivariate analysis, when adjusted for biopsy Gleason score, age, PSA, and ECOG PS (all at time of blood draw), only high plasma IL-8 (on a continuous basis) was significantly associated with reduced OS (HR = 1.43; p = 0.019). Conclusions: In mCRPC patients, high plasma IL-8 and PD-L1 levels were associated with reduced OS (separately and combined). Circulating IL-8 and PD-L1 evaluation may inform prognosis in mCRPC and could be considered as biomarkers in future studies determining response to immune checkpoint inhibitor and anti-IL8 therapy.

Notch signaling is an evolutionarily conserved pathway associated with the development and differentiation of all metazoans. It is needed for proper germ layer formation and segmentation of the embryo and controls the timing and duration of differentiation events in a dynamic manner. Perturbations of Notch signaling result in blockades of developmental cascades, developmental anomalies, and cancers. An in-depth understanding of Notch signaling is thus required to comprehend the basis of development and cancer, and can be further exploited to understand and direct the outcomes of targeted cellular differentiation into desired cell types and complex tissues from pluripotent or adult stem and progenitor cells. In this chapter, we briefly summarize the molecular, evolutionary, and developmental basis of Notch signaling. We will focus on understanding the basics of Notch signaling and its signaling control mechanisms, its developmental outcomes and perturbations leading to developmental defects, as well as have a brief look at mutations of the Notch signaling pathway causing human hereditary disorders or cancers.

1031 Background: In MA.31 the trastuzumab-taxane combination led to longer PFS than lapatinib-taxane in HER2+ metastatic breast cancer (MBC). In MA.31 we previously reported the prognostic/predictive utility of pretreatment serum PD-L1 (SABCS 2018, PD3-10) and serum activin A (SABCS 2016, P6-07-06) separately; here we evaluate them combined. Methods: MA.31 accrued 652 centrally and/or locally-identified HER2-positive patients, and pretreatment serum was available for 382 patients (184 in trastuzumab arm, 198 in lapatinib arm). The ELLA immunoassay platform (ProteinSimple, San Jose, CA) was used to quantitate serum PD-L1, and ELISA for activin A (R&D Systems, Minneapolis, MN). Results: In correlation analysis, pretreatment serum PD-L1 was moderately correlated with serum activin A (r = 0.21, p = 0.004). In univariate analysis for OS, the combination of higher serum PD-L1 and higher serum activin A (median cutpoints) (vs. both low) was significant for shorter OS in the trastuzumab arm (HR 6.62, p=0.0005) and in the lapatinib arm (HR 3.25, p=0.0003)(table). In multivariate analysis for OS (17 covariates included), elevated serum activin A/PD-L1 combination remained the most significant independent covariate in the trastuzumab arm (HR 12.40, p=0.001), and in the lapatinib arm (HR 5.2, p=0.0001). Conclusions: In the CCTG MA.31 trial, elevated pretreatment serum activin A (TGF-B superfamily) and PD-L1 was associated with a shorter OS to HER2-targeted treatment. Multiple mechanisms, including immune evasion, may decrease the effectiveness of HER2-targeted therapy. Elevated serum activin A and PD-L1 may identify HER2-positive MBC patients who would benefit from inhibitors of the HER2, PD-1, and activin A pathways. [Table: see text]

4131 Background: We previously reported the prognostic and predictive utility of pretreatment serum PD-L1 in the CCTG MA.31 serum bank (SABCS 2018, abstr PD3-10). IL-8 (CXCL8) is a pro-inflammatory cytokine that binds to CXCR1 and CXCR2 and promotes tumor immune escape and progression. High serum IL-8 levels are associated with poor prognosis in many cancers, and have recently been reported to predict for reduced OS to nivolumab in lung cancer and melanoma (ASCO 2018, abstr #3025). In this study, we retrospectively evaluated combined pretreatment serum IL-8 and PD-L1 on overall survival (OS) from a phase III randomized pancreatic cancer trial of first-line therapy (octreotide + 5-FU vs. 5-FU) that had reported no significant OS difference between treatment arms. Methods: This study had 147 patients with serum available for this retrospective biomarker analysis from an advanced pancreatic cancer phase III clinical trial.TheELLA immunoassay platform (ProteinSimple, San Jose, CA) was utilized to quantitate serum levels of IL-8 and PD-L1. Kaplan-Meier life table analysis was used to correlate serum biomarkers with overall survival (OS). Results: In univariate analysis, pretreatment serum IL-8 was a significant biomarker as a continuous variable (HR = 1.004; p = 0.012) and trended significant at the median cutpoint (HR = 1.379; p = 0.098) for OS, however serum PD-L1 was not significant at any cutpoint. When serum PD-L1 and IL-8 levels were analyzed as combined biomarkers (median cutpoints), the serum IL-8 high / PD-L1 high cohort had a significantly shorter OS vs the serum IL-8 low / PD-L1 low cohort (HR = 1.816; p = 0.017). Conclusions: In this phase III randomized clinical trial in advanced pancreatic cancer, pretreatment serum IL-8 was a significant biomarker for OS, but serum PD-L1 was not. Higher combined pretreatment serum levels of PD-L1 and IL-8 (both biomarkers high vs. both low) were prognostic for reduced OS in this phase III pancreatic cancer trial. Further study of circulating IL-8 and PD-L1 is warranted in pancreatic cancer for evaluation of targeted and investigational therapies, including the immune checkpoint inhibitors and anti-IL8 therapy.

ProblemQuantitative measurement of proteins in bodily fluids or cellular preparations is critical for the evaluation of biomarkers or the study of complex cellular processes. While immunoassays are the most common quantitative approach used so far, they are not practical for the evaluation of multiple proteins. Microfluidic technology allows a fine spatial control in immobilizing proteins and biomolecules inside microchannels, eliminating cross‐reactivity between competing analytes, and allowing rapid and sensitive detection of targeted antigens for multiple applications. We report the characterization and validation of the Simple Plex™ platform for the detection and quantification of cytokines and chemokines from human and mouse samples.MethodCytokine and chemokine expression levels were determined using Simple Plex cartridges from ProteinSimple. Serum samples were obtained from the Yale Biorepository.ResultsOur data demonstrate an excellent correlation between the results obtained with Simple Plex and conventional immunoassays such as ELISA and Luminex.ConclusionWe describe the characterization and validation of Simple Plex, a novel multi‐analyte, automated microfluidic platform that allows the evaluation of cytokines and chemokines from human and mice biological samples. Simple Plex showed significant advantages over traditional approaches in terms of low sample volume requirements, sensitivity and dynamic range, coefficient of variation, and reproducibility.