Recent advances in upstream process development for production of recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in-vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes current available information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in-depth understanding of production kinetics and quality control.

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step.

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N-1 seed (Process B), by perfusion N-1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C-terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C-terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next-generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge-based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next-generation continuous bioprocessing, it is essential to embrace a fundamental shift-in-paradigm from "quality-by-testing" to "quality-by-design," which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Technology outlook for real-time quality attribute and process parameter monitoring in biopharmaceutical development-A review.

Real-time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real-time release (RTR) of biopharmaceuticals. A well-defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real-time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated-data piping, analysis, aggregation, visualization, and smart utility of data for advanced-data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated-sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced-analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Hypoxia and transforming growth factor-beta1 pathway activation promote Chinese Hamster Ovary cell aggregation.

Suspension cultivation is the preferred mode of operation for the large-scale production of many biologics. Chinese Hamster Ovary (CHO) cells are anchorage-dependent in origin, but they have been widely adapted to suspension culture. In suspension culture, formation of CHO cell aggregates is a common phenomenon and compromises cell culture performance in multiple ways. To better understand the underlying mechanisms that regulate cell aggregation, we utilized CHO-specific transcriptome profiling as a screening tool and demonstrated that many genes encoding extracellular matrix (ECM) proteins were upregulated in the cultures with increased cell aggregation. Significantly, hypoxia was identified to be a cause for promoting CHO cell aggregation, and transforming growth factor beta1 (TGFß1) pathway activation served as an intermediate step mediating this biological cascade. These transcriptomics findings were confirmed by cell culture experiments, and it was further demonstrated that adding recombinant TGFß1 to the culture significantly increased ECM protein fibronectin expression and cell aggregation. The results of this study emphasize the importance of adequate mixing and oxygen supply for suspension cultures from a new angle, and regulating the TGFß1 pathway is proposed as a new strategy for mitigating cell aggregation to improve cell culture performance.

Early identification of unusually clustered mutations and root causes in therapeutic antibody development.

This study reports findings of an unusual cluster of mutations spanning 22 bp (base pairs) in a monoclonal antibody expression vector. It was identified by two orthogonal methods: mass spectrometry on expressed protein and next-generation sequencing (NGS) on the plasmid DNA. While the initial NGS analysis confirmed the designed sequence modification, intact mass analysis detected an additional mass of the antibody molecule expressed in CHO cells. The extra mass was eventually found to be associated with unmatched nucleotides in a distal region by checking full-length sequence alignment plots. Interestingly, the complementary sequence of the mutated sequence was a reverse sequence of the original sequence and flanked by two 10-bp reverse-complementary sequences, leading to an undesirable DNA recombination. The finding highlights the necessity of rigorous examination of expression vector design and early monitoring of molecule integrity at both DNA and protein levels to prevent clones from having sequence variants during cell line development.

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose ß-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose ß-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.

A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors.

Control of carbon dioxide within the optimum range is important in mammalian bioprocesses at the manufacturing scale in order to ensure robust cell growth, high protein yields, and consistent quality attributes. The majority of bioprocess development work is done in laboratory bioreactors, in which carbon dioxide levels are more easily controlled. Some challenges in carbon dioxide control can present themselves when cell culture processes are scaled up, because carbon dioxide accumulation is a common feature due to longer gas-residence time of mammalian cell culture in large scale bioreactors. A carbon dioxide stripping model can be used to better understand and optimize parameters that are critical to cell culture processes at the manufacturing scale. The prevailing carbon dioxide stripping models in literature depend on mass transfer coefficients and were applicable to cell culture processes with low cell density or at stationary/cell death phase. However, it was reported that gas bubbles are saturated with carbon dioxide before leaving the culture, which makes carbon dioxide stripping no longer depend on a mass transfer coefficient in the new generation cell culture processes characterized by longer exponential growth phase, higher peak viable cell densities, and higher specific production rate. Here, we present a new carbon dioxide stripping model for manufacturing scale bioreactors, which is independent of carbon dioxide mass transfer coefficient, but takes into account the gas-residence time and gas CO2 saturation time. The model was verified by CHO cell culture processes with different peak viable cell densities (7 to 12 × 106 cells mL-1 ) for two products in 5,000-L and 25,000-L bioreactors. The model was also applied to a next generation cell culture process to optimize cell culture conditions and reduce carbon dioxide levels at manufacturing scale. The model provides a useful tool to understand and better control cell culture carbon dioxide profiles for process development, scale up, and characterization. Biotechnol. Bioeng. 2017;114: 1184-1194. © 2016 Wiley Periodicals, Inc.

The use of 'Omics technology to rationally improve industrial mammalian cell line performance.

Biologics represent an increasingly important class of therapeutics, with 7 of the 10 top selling drugs from 2013 being in this class. Furthermore, health authority approval of biologics in the immuno-oncology space is expected to transform treatment of patients with debilitating and deadly diseases. The growing importance of biologics in the healthcare field has also resulted in the recent approvals of several biosimilars. These recent developments, combined with pressure to provide treatments at lower costs to payers, are resulting in increasing need for the industry to quickly and efficiently develop high yielding, robust processes for the manufacture of biologics with the ability to control quality attributes within narrow distributions. Achieving this level of manufacturing efficiency and the ability to design processes capable of regulating growth, death and other cellular pathways through manipulation of media, feeding strategies, and other process parameters will undoubtedly be facilitated through systems biology tools generated in academic and public research communities. Here we discuss the intersection of systems biology, 'Omics technologies, and mammalian bioprocess sciences. Specifically, we address how these methods in conjunction with traditional monitoring techniques represent a unique opportunity to better characterize and understand host cell culture state, shift from an empirical to rational approach to process development and optimization of bioreactor cultivation processes. We summarize the following six key areas: (i) research applied to parental, non-recombinant cell lines; (ii) systems level datasets generated with recombinant cell lines; (iii) datasets linking phenotypic traits to relevant biomarkers; (iv) data depositories and bioinformatics tools; (v) in silico model development, and (vi) examples where these approaches have been used to rationally improve cellular processes. We critically assess relevant and state of the art research being conducted in academic, government and industrial laboratories. Furthermore, we apply our expertise in bioprocess to define a potential model for integration of these systems biology approaches into biologics development.

Validation of a CFD model for cell culture bioreactors at large scale and its application in scale-up.

Among all the operating parameters that control the cell culture environment inside bioreactors, appropriate mixing and aeration are crucial to ensure sufficient oxygen supply, homogeneous mixing, and CO2 stripping. A model-based manufacturing facility fit approach was applied to define agitation and bottom air flow rates during the process scale-up from laboratory to manufacturing, of which computational fluid dynamics (CFD) was the core modeling tool. The realizable k-ε turbulent dispersed Eulerian gas-liquid flow model was established and validated using experimental values for the volumetric oxygen transfer coefficient (kLa). Model validation defined the process operating parameter ranges for application of the model, identified mixing issues (e.g., impeller flooding, dissolved oxygen gradients, etc.) and the impact of antifoam on kLa. Using the CFD simulation results as inputs to the models for oxygen demand, gas entrance velocity, and CO2 stripping aided in the design of the agitation and bottom air flow rates needed to meet cellular oxygen demand, control CO2 levels, mitigate risks for cell damage due to shear, foaming, as well as fire hazards due to high O2 levels in the bioreactor gas outlet. The recommended operating conditions led to the completion of five manufacturing runs with a 100% success rate. This model-based approach achieved a seamless scale-up and reduced the required number of at-scale development batches, resulting in cost and time savings of a cell culture commercialization process.

Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media.

Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40-80°C) and pH (7.6-10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.

Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing.

Core fucosylation is a highly prevalent and significant feature of N-glycosylation in therapeutic monoclonal antibodies produced by mammalian cells where its absence (afucosylation) plays a key role in treatment safety and efficacy. Notably, even slight changes in the level of afucosylation can have a considerable impact on the antibody-dependent cell-mediated cytotoxicity. Therefore, implementing control over afucosylation levels is important in upstream manufacturing to maintain consistent quality across batches of product, since standard downstream processing does not change afucosylation. In this review, the influences and strategies to control afucosylation are presented. In particular, there is emphasis on upstream manufacturing culture parameters and media supplementation, as these offer particular advantages as control strategies over alternative approaches such as cell line engineering and chemical inhibitors. The review discusses the relationship between the afucosylation influences and the underlying cellular metabolism to promote increased process understanding. Also, briefly highlighted is the value of empirical and mechanistic models in evaluating and designing control methods for core fucosylation.

Antibody charge variant modulation by in vitro enzymatic treatment in different Chinese hamster ovary cell cultures.

Charge variants represent a critical quality attribute that must be controlled during the development and manufacturing of monoclonal antibodies (mAb). Previously, we reported the development of a cost-effective enzymatic treatment capable of removing the C-terminal lysine from a mAb produced by a Chinese hamster ovary (CHO) GS cell line. This treatment resulted in a significant decrease in basic charge variants and a corresponding improvement in the main peak, enabling a longer cell culture production duration for titer improvement. Here, we describe this enzymatic treatment protocol in detail and demonstrate its applicability to two additional mAbs produced by distinct industrial cell lines. The simple addition of carboxypeptidase B (CpB) at a ratio of 1:10,000 (w/w) to whole cell cultures significantly improved the main peaks for both mAbs without affecting other critical quality attributes, including size exclusion chromatography impurities and N-glycans. Our results demonstrate that this in vitro CpB treatment protocol can be used as a platform strategy to improve main peak for mAbs that exhibit high levels of basic variants attributable to C-terminal lysines. An in vitro enzymatic treatment in general may be another good addition to existing in vivo CHO cell culture strategies for titer improvement and control of critical quality attributes.

Upstream cell culture process characterization and in-process control strategy development at pandemic speed.

As of early 2022, the coronavirus disease 2019 (COVID-19) pandemic remains a substantial global health concern. Different treatments for COVID-19, such as anti-COVID-19 neutralizing monoclonal antibodies (mAbs), have been developed under tight timelines. Not only mAb product and clinical development but also chemistry, manufacturing, and controls (CMC) process development at pandemic speed are required to address this highly unmet patient need. CMC development consists of early- and late-stage process development to ensure sufficient mAb manufacturing yield and consistent product quality for patient safety and efficacy. Here, we report a case study of late-stage cell culture process development at pandemic speed for mAb1 and mAb2 production as a combination therapy for a highly unmet patient treatment. We completed late-stage cell culture process characterization (PC) within approximately 4 months from the cell culture process definition to the initiation of the manufacturing process performance qualification (PPQ) campaign for mAb1 and mAb2, in comparison to a standard one-year PC timeline. Different strategies were presented in detail at different PC steps, i.e., pre-PC risk assessment, scale-down model development and qualification, formal PC experiments, and in-process control strategy development for a successful PPQ campaign that did not sacrifice quality. The strategies we present may be applied to accelerate late-stage process development for other biologics to reduce timelines.

N-1 Perfusion Platform Development Using a Capacitance Probe for Biomanufacturing.

Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO) cells can be achieved by implementing perfusion operation at the N-1 stage for biomanufacturing. N-1 perfusion seed with much higher final viable cell density (VCD) than a conventional N-1 batch seed can be used to significantly increase the inoculation VCD for the subsequent fed-batch production (referred as N stage), which results in a shorter cell growth phase, higher peak VCD, or higher titer. In this report, we incorporated a process analytical technology (PAT) tool into our N-1 perfusion platform, using an in-line capacitance probe to automatically adjust the perfusion rate based on real-time VCD measurements. The capacitance measurements correlated linearly with the offline VCD at all cell densities tested (i.e., up to 130 × 106 cells/mL). Online control of the perfusion rate via the cell-specific perfusion rate (CSPR) decreased media usage by approximately 25% when compared with a platform volume-specific perfusion rate approach and did not lead to any detrimental effects on cell growth. This PAT tool was applied to six mAbs, and a platform CSPR of 0.04 nL/cell/day was selected, which enabled rapid growth and maintenance of high viabilities for four of six cell lines. In addition, small-scale capacitance data were used in the scaling-up of N-1 perfusion processes in the pilot plant and in the GMP manufacturing suite. Implementing a platform approach based on capacitance measurements to control perfusion rates led to efficient process development of perfusion N-1 for supporting high-density CHO cell cultures for the fed-batch process intensification.

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

Real time monitoring of multiple parameters in mammalian cell culture bioreactors using an in-line Raman spectroscopy probe.

The FDA's process analytical technology initiative encourages drug manufacturers to apply innovative ideas to better understand their processes. There are many challenges to applying these techniques to monitor mammalian cell culture bioreactors for biologics manufacturing. These include the ability to monitor multiple components in complex medium formulations non-invasively and in-line. We report results that demonstrate, for the first time, the technical feasibility of the in-line application of Raman spectroscopy for monitoring a mammalian cell culture bioreactor. A Raman probe was used for the simultaneous prediction of culture parameters including glutamine, glutamate, glucose, lactate, ammonium, viable cell density, and total cell density.

High throughput screening data for a case study of CHO cell culture process development.

In this article, we present four sets of data from high-throughput screening (HTS) studies of different chemically defined media using an industrially relevant Chinese hamster ovary (CHO) cell line. While complex hydrolysate media was used in the early phase process development and manufacturing of a monoclonal antibody (mAb), here we seek to determine an appropriate chemically defined media for late phase process development. Over 150 combinations of chemically defined basal media, feed media, and basal and feed media supplements, such as polyphenolic flavonoid antioxidants (including rosmarinic acid (RA)), were evaluated in four HTS studies to replace the complex hydrolysate media. Specifically, these four screening studies incorporated custom design of experiment (DOE), one-factor-at-a-time (OFAT), and definitive screening design methodologies for titer improvement. Titer was improved two fold compared to the early phase process using the addition of RA to chemically defined media. This dataset exemplifies how HTS can be used as an effective approach to systematically and statistically determine media composition and supplementation to increase mAb titer. These data were presented in connection with a published paper [1].

Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells.

CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.