A fast and simple approach to optimize the unit operation high pressure homogenization - a case study for a soluble therapeutic protein in E. coli

Escherichia coli is one of the most commonly used host organisms for the production of recombinant biopharmaceuticals. E. coli is usually characterized by fast growth on cheap media and high productivity, but one drawback is its intracellular product formation. Product recovery from E. coli bioprocesses requires tedious downstream processing (DSP). A typical E. coli DSP for an intracellular product starts with a cell disruption step to access the product. Different methods exist, but a scalable process is usually achieved by high pressure homogenization (HPH). The protocols for HPH are often applied universally without adapting them to the recombinant product, even though HPH can affect product quantity and quality. Based on our previous study on cell disruption efficiency, we aimed at screening operational conditions to maximize not only product quantity, but also product quality of a soluble therapeutic protein expressed in E. coli. We screened for critical process parameters (CPPs) using a multivariate approach (design of experiments; DoE) during HPH to maximize product titer and achieve sufficient product quality, based on predefined critical quality attributes (CQAs). In this case study, we were able to gain valuable knowledge on the efficiency of HPH on E. coli cell disruption, product release and its impact on CQAs. Our results show that HPH is a key unit operation that has to be optimized for each product.     

Identification and classification of host cell proteins during biopharmaceutical process development

As significant improvements in volumetric antibody productivity have been achieved by advances in upstream processing over the last decade, and harvest material has become progressively more difficult to recover with these intensified upstream operations, the segregation of upstream and downstream processing has remained largely unchanged. By integrating upstream and downstream process development, product purification issues are given consideration during the optimization of upstream operating conditions, which mitigates the need for extensive and expensive clearance strategies downstream. To investigate the impact of cell culture duration on critical quality attributes, CHO-expressed IgG1 was cultivated in two 2 L bioreactors with samples taken on days 8, 10, 13, 15, and 17. The material was centrifuged, filtered and protein A purified on a 1 ml HiTrap column. Host cell protein (HCP) identification by mass spectrometry (MS) was applied to this system to provide insights into cellular behavior and HCP carryover during protein A purification. It was shown that as cultivation progressed from day 8 to 17 and antibody titer increased, product quality declined due to an increase in post-protein A HCPs (from 72 to 475 peptides detected by MS) and a decrease in product monomer percentage (from 98% to 95.5%). Additionally, the MS data revealed an increase in the abundance of several classes of post-protein A HCPs (e.g., stress response proteins and indicators of cell age), particularly on days 15 and 17 of culture, which were associated with significant increases in total overall HCP levels. This provides new insight into the specific types of HCPs that are retained during mAb purification and may be used to aid process development strategies.     

Integrated process development—a robust,rapid method for inclusion body harvesting and processing at the microscale level

Escherichia coli stores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a “design of experiments” study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples.     

Scale‐down of the inactivated polio vaccine production process

The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production processes is necessary as the initial process development has been done decades ago. An up‐to‐date lab‐scale version encompassing the legacy inactivated polio vaccine production process was set‐up. This lab‐scale version should be representative of the large scale, meaning a scale‐down model, to allow experiments for process optimization that can be readily applied. Initially the separate unit operations were scaled‐down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large‐scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small‐scale. Challenges in translating large‐scale operations to lab‐scale are discussed, and the concessions that needed to be made are described. The current scale‐down model for cell and virus culture (2.3‐L) presents a feasible model with its production scale counterpart (750‐L) when operated at setpoint. Also, the current scale‐down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small‐scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it is shown that the scale‐down unit operations can be used consecutively to prepare trivalent vaccine at lab‐scale with comparable characteristics to the product produced at manufacturing scale. Biotechnol. Bioeng. 2013; 110: 1354–1365. © 2012 Wiley Periodicals, Inc.     

A new flow cell and chemometric protocol for implementing in-line Raman spectroscopy in chromatography

On-line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in-line flow cell with a reduced dead volume of 80 μL and a reflector to increase the signal intensity as well as developing a chemometric modeling path. In this context, measurement settings were adjusted and spectra were taken from different chromatographic breakthrough curves of IgG1 in harvest. The resulting models show a small average RMSEP of 0.12 mg/mL, on a broad calibration range from 0 to 2.82 mg/mL IgG1. This work highlights the benefits of model assisted Raman spectroscopy in chromatography with complex backgrounds, lays the fundamentals for in-line monitoring of IgG1, and enables advanced control strategies. Moreover, the approach might be extended to further critical quality attributes like aggregates or could be transferred to other process steps.     

Differential response in downstream processing of CHO cells grown under mild hypothermic conditions

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L^?1. However, host cell proteins, measured by ELISA, increased by ～50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI‐TOF MS and 2D‐PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013     

Biomanufacturing process analytical technology (PAT) application for downstream processing: Using dissolved oxygen as an indicator of product quality for a protein refolding reaction

Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small‐scale bioreactor (3 L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse‐phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (k_La) is used for scale‐up of the aeration parameters to manufacturing scale tanks (2,000 L, 15,000 L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to “actively manage process variability using a knowledge‐based approach.” Biotechnol. Bioeng. 2009; 104: 340–351 © 2009 Wiley Periodicals, Inc.     

Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance

During early stage bioprocess development, characterizing interactions between unit operations is a key challenge. Such interactions include the release of host cell enzymes early in the process causing losses in product quality downstream. Using a CHO-expressed IgG1 system, the impact of cell culture duration was investigated using a 50 L bioreactor and performing scale-down protein A purification. While antibody titer doubled during the last week of culture, the post-protein A host cell protein (HCP) levels increased from 243 to 740 ppm. Effects of pH and temperature were then explored using fed-batch ambr250 bioreactors, and parameters enabling higher titers were linked to a decrease in post-protein A product purity. These trade-offs between titer and product quality were visualized using a window of operation. The downstream space was explored further by exposing shake flask material to shear representative of disc stack centrifugation, prior to purification, and by adding polishing chromatography. While product quality decreased with progressing cultivation, cells became more shear resistant. Polishing chromatography resulted in product fragmentation which increased fourfold from Day 10 to 24, adding constraint to achieving both efficient HCP clearance as well as high monomer purities. These examples highlight the importance of adopting integrated approaches to upstream and downstream development strategies to enable whole process optimization.     

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D‐PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post‐protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D‐PAGE can be used for monitoring and identification of HCPs post‐protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell‐engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251. © 2012 Wiley Periodicals, Inc.     

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

Non‐mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof‐of‐concept for developing affinity peptide‐based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987–998, 2018     

Intracellular response to process optimization and impact on productivity and product aggregates for a high‐titer CHO cell process

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in‐depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra‐ and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.     

A dynamic model linking cell growth to intracellular metabolism and extracellular by-product accumulation

Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies.     

Kinetic model for biotransformation of digitoxin in plant cell suspension culture ofDigitalis lanata

Batch suspension cultures ofDigitalis lanata plant cell were performed to investigate the biotransformation of digitoxin.Digitalis lanata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to describe cell growth, substrate consumption, depletion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and formation of deacetyllanatoside C. The digoxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately predict cell growth, substrate consumption and product synthesis. And it can provide a useful framework for quantitative analysis of biotransformation in a plant cell culture system.     

Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.     

Ubiquitin-specific protease 7 (USP7) is essential for endometrial stromal cell decidualization in mice

Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating (DUB) enzyme family, regulates protein stability and has a well-characterized function in tumorigenesis. Given its critical role in growth and development, it was speculated to be involved in modulating processes in the female reproductive system but its exact role has not been elucidated. Decidualization is one of the key processes in pregnancy and aberrant decidualization is a cause of pregnancy failure. The uterine endometrium layer undergoes significant structural and functional changes during decidualization in preparation for and after embryo implantation. Here, we hypothesized that USP7 could be involved in mediating endometrial stromal cell (ESC) decidualization and set out to determine its function with a primary stromal cell culture. Using in situ hybridization and immunohistochemical techniques, we observed increased USP7 expression during uterine decidualization and found that it was predominantly localized to the decidual zone in the post-implantation uterus. Since the ovarian hormones, progesterone (P4) and estrogen (E2), function in promoting stroma decidualization, we investigated their relationship with USP7 expression and found that they exert minimal influence. Moreover, increased USP7 expression observed during deciduoma development was found to be independent of blastocyst attachment. Using a specific USP7 inhibitor, HBX19818, we demonstrated an additional novel role for USP7 in endometrial stroma decidualization in mice during early pregnancy. Our findings could potentially be applied towards future research and development in female infertility.     

泛素化特异性酶（USP2a）与肿瘤发生的相关分子机制

去泛素化酶USP2a是去泛素化酶家族(DUBs)的一个成员,为半胱氨酸蛋白酶,是一种重要的特异性去泛素化水解酶。USP2a具有结构和功能多样性,其结构多样化使得这些酶具有一些特异性作用靶点,特别是在基因表达调控中靶向的生理底物种类繁多。特异性蛋白泛素化水平的动态变化涉及到基因表达活化和失活的多种机制以及信号通路转导的多个环节。越来越多的文献报道了去泛素化酶相互作用网络的组成及其重要性。USP2a调节多种重要的细胞生长和分化调节因子及信号转导因子的稳定性和功能,通过USP2a的去泛素化作用以及诱导它们之间相互反应对机体进行相应调控,特别是在调控转录因子、细胞周期和细胞凋亡自噬上发挥重要作用。USP2a的过表达在体内外都表现出致癌性,其靶蛋白通过各种途径影响肿瘤发生发展。通过对人类肿瘤发生发展的相关分子机制及信号通路影响的深入研究,USP2a有望成为肿瘤治疗的新靶点。现就去泛素化酶与人类肿瘤发生发展的相关分子机制及该领域的研究进展作一综述。     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.