Search results for: asymptotic complexity

Authors: Rowan Moore, Kai Scheffler, Eugen Damoc, Jennifer Sutton, Aaron Bailey, Stephane Houel, Simon Cubbon, Jonathan Josephs

Abstract:

Purpose: MS analysis of monoclonal antibodies (mAbs) at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to various modifications which may affect efficacy. Intact proteins analyzed by MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or native-like conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Methods: Three commercially available mAbs were used for all experiments. Intact proteins were desalted online using size exclusion chromatography (SEC) or reversed phase chromatography coupled on-line with a mass spectrometer. For streamlined use of the LC- MS platform we used a single SEC column and alternately selected specific mobile phases to perform separations in either denaturing or native-like conditions: buffer A (20 % ACN, 0.1 % FA) with Buffer B (100 mM ammonium acetate). For peptide analysis mAbs were proteolytically digested with and without prior reduction and alkylation. The mass spectrometer used for all experiments was a commercially available Thermo Scientific™ hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with the new BioPharma option which includes a new High Mass Range (HMR) mode that allows for improved high mass transmission and mass detection up to 8000 m/z. Results: We have analyzed the profiles of three mAbs under reducing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The presence of high salt under denaturing conditions was found to influence the observed charge state envelope and impact mass accuracy after spectral deconvolution. The significantly lower charge states observed under native conditions improves the spatial resolution of protein signals and has significant benefits for the analysis of antibody mixtures, e.g. lysine variants, degradants or sequence variants. This type of analysis requires the detection of masses beyond the standard mass range ranging up to 6000 m/z requiring the extended capabilities available in the new HMR mode. We have compared each antibody sample that was analyzed individually with mixtures in various relative concentrations. For this type of analysis, we observed that apparent native structures persist and ESI is benefited by the addition of low amounts of acetonitrile and formic acid in combination with the ammonium acetate-buffered mobile phase. For analyses on the peptide level we analyzed reduced/alkylated, and non-reduced proteolytic digests of the individual antibodies separated via reversed phase chromatography aiming to retrieve as much information as possible regarding sequence coverage, disulfide bridges, post-translational modifications such as various glycans, sequence variants, and their relative quantification. All data acquired were submitted to a single software package for analysis aiming to obtain a complete picture of the molecules analyzed. Here we demonstrate the capabilities of the mass spectrometer to fully characterize homogeneous and heterogeneous therapeutic proteins on one single platform. Conclusion: Full characterization of heterogeneous intact protein mixtures by improved mass separation on a quadrupole-Orbitrap™ mass spectrometer with extended capabilities has been demonstrated.

Keywords: disulfide bond analysis, intact analysis, native analysis, mass spectrometry, monoclonal antibodies, peptide mapping, post-translational modifications, sequence variants, size exclusion chromatography, therapeutic protein analysis, UHPLC

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Authors: Reza Nematirad, Anil Pahwa, Balasubramaniam Natarajan

Abstract:

Time series data consist of successive data points collected over a period of time. Accurate prediction of future values is essential for informed decision-making in several real-world applications, including electricity load demand forecasting, lifetime estimation of industrial machinery, traffic planning, weather prediction, and the stock market. Due to their critical relevance and wide application, there has been considerable interest in time series forecasting in recent years. However, the proliferation of sensors and IoT devices, real-time monitoring systems, and high-frequency trading data introduce significant intricate temporal variations, rapid changes, noise, and non-linearities, making time series forecasting more challenging. Classical methods such as Autoregressive integrated moving average (ARIMA) and Exponential Smoothing aim to extract pre-defined temporal variations, such as trends and seasonality. While these methods are effective for capturing well-defined seasonal patterns and trends, they often struggle with more complex, non-linear patterns present in real-world time series data. In recent years, deep learning has made significant contributions to time series forecasting. Recurrent Neural Networks (RNNs) and their variants, such as Long short-term memory (LSTMs) and Gated Recurrent Units (GRUs), have been widely adopted for modeling sequential data. However, they often suffer from the locality, making it difficult to capture local trends and rapid fluctuations. Convolutional Neural Networks (CNNs), particularly Temporal Convolutional Networks (TCNs), leverage convolutional layers to capture temporal dependencies by applying convolutional filters along the temporal dimension. Despite their advantages, TCNs struggle with capturing relationships between distant time points due to the locality of one-dimensional convolution kernels. Transformers have revolutionized time series forecasting with their powerful attention mechanisms, effectively capturing long-term dependencies and relationships between distant time points. However, the attention mechanism may struggle to discern dependencies directly from scattered time points due to intricate temporal patterns. Lastly, Multi-Layer Perceptrons (MLPs) have also been employed, with models like N-BEATS and LightTS demonstrating success. Despite this, MLPs often face high volatility and computational complexity challenges in long-horizon forecasting. To address intricate temporal variations in time series data, this study introduces Times2D, a novel framework that parallelly integrates 2D spectrogram and derivative heatmap techniques. The spectrogram focuses on the frequency domain, capturing periodicity, while the derivative patterns emphasize the time domain, highlighting sharp fluctuations and turning points. This 2D transformation enables the utilization of powerful computer vision techniques to capture various intricate temporal variations. To evaluate the performance of Times2D, extensive experiments were conducted on standard time series datasets and compared with various state-of-the-art algorithms, including DLinear (2023), TimesNet (2023), Non-stationary Transformer (2022), PatchTST (2023), N-HiTS (2023), Crossformer (2023), MICN (2023), LightTS (2022), FEDformer (2022), FiLM (2022), SCINet (2022a), Autoformer (2021), and Informer (2021) under the same modeling conditions. The initial results demonstrated that Times2D achieves consistent state-of-the-art performance in both short-term and long-term forecasting tasks. Furthermore, the generality of the Times2D framework allows it to be applied to various tasks such as time series imputation, clustering, classification, and anomaly detection, offering potential benefits in any domain that involves sequential data analysis.

Keywords: derivative patterns, spectrogram, time series forecasting, times2D, 2D representation

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