Host Cell Proteins Research Articles

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1–2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.

Engineering protein A ligands to mitigate antibody loss during high-pH washes in protein A chromatography

Protein A chromatography is a workhorse in monoclonal antibody (mAb) manufacture since it provides effective separation of mAbs from impurities such as host-cell proteins (HCPs) in a single capture step. HCP clearance can be aided by the inclusion of a wash step prior to low-pH elution. Although high-pH washes can be effective in removing additional HCPs from the loaded column, they may also contribute to a reduced mAb yield. In this work we show that this yield loss is reflected in a pH-dependent variation of the equilibrium binding capacity of the protein A resin, which is also observed for the capacity of the Fc fragments alone and therefore not a result of steric interactions involving the Fab fragments in the intact mAbs. We therefore hypothesized that the high-pH wash loss was due to protonation or deprotonation of ionizable residues on the protein A ligand. To evaluate this, we applied a rational protein engineering approach to the Z domain (the Fc-binding component of most commercial protein A ligands) and expressed engineered mutants in E. coli. Biolayer interferometry and affinity chromatography experiments showed that some of the Z domain mutants were able to mitigate wash loss at high pH while maintaining similar binding characteristics at neutral pH. These experiments enabled elucidation of the roles of specific interactions in the Z domain - Fc complex, but more importantly offer a route to ameliorating the disadvantages of high-pH washes in protein A chromatography.

Minimal purification method enables developability assessment of recombinant proteins.

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii.

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry

Monitoring of host cell proteins (HCPs) during the manufacturing of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug products. Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. Here, we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process. While we also explored spectral library-free DIA interpretation, the spectral library-based approach still showed highest accuracy and reproducibility (coefficients of variation < 10%) with a sensitivity down to the sub-ng/mg mAb level. Thus, this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Comparison of host cell protein removal by depth filters with diatomaceous earth and synthetic silica filter aids using model proteins.

A number of studies have demonstrated that depth filtration can provide significant adsorptive removal of host cell proteins (HCP), but there is still considerable uncertainty regarding the underlying factors controlling HCP binding. This study compared the binding characteristics of two fine grade depth filters, the X0SP (polyacrylic fiber with a synthetic silica filter aid) and X0HC (cellulose fibers with diatomaceous earth (DE) as a filter aid), using a series of model proteins with well-defined physical characteristics. Protein binding to the X0SP filter was dominated by electrostatic interactions with greatest capacity for positively-charged proteins. In contrast, the X0HC filter showed greater binding of more hydrophobic proteins although electrostatic interactions also played a role. In addition, ovotransferrin showed unusually high binding capacity to the X0HC, likely due to interactions with metals in the DE. Scanning Electron Microscopy with Energy Dispersive Spectroscopy was used to obtain additional understanding of the binding behavior. These results provide important insights into the physical phenomena governing HCP binding to both fully synthetic and natural (cellulose + DE) depth filters.

The deubiquitylase-ubiquitin-specific protease 4 absence in HeLa cells leads to a reduction in Semliki Forest virus replication

The emergence of a potential epidemic of alphaviruses is a growing concern for public health officials and the global community. These highly contagious and fast-spreading viruses have the potential to cause severe illness, disability, and even death, and are a growing threat to populations worldwide. Understanding the interaction between viruses, deubiquitylases (DUBs), and other human host cell proteins during replication is crucial when developing targeted antiviral medications. Many viruses have developed strategies to manipulate the host ubiquitin system by hijacking DUBs to evade the host immune response and promote viral replication, making DUBs attractive therapeutic targets. Small interfering RNA (siRNA) knockdown of USP4 expression inhibited Semliki Forest virus (SFV) replication and reduced virus titre. These findings highlight the potential to target USP4 as an antiviral treatment during alphavirus infection.

Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics.

Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.

Integrated double flow-through purification of monoclonal antibodies using membrane adsorbers and single-pass tangential flow filtration

A flow-through process for monoclonal antibody (mAb) purification has been created by integrating two different membrane adsorbers (MAs) with single-pass tangential flow filtration (SPTFF) for continuous buffer exchange. First, a design of experiments (DoE) was carried out to investigate the influence of pH and conductivity on the removal of desoxyribonucleic acid (DNA) and host cell proteins (HCP), and to select buffer conditions for mAb flow-through mode operation for four different MAs. As next, breakthrough curve experiments were performed with the selected buffer conditions for each MA to analyze the binding behavior of mAb, DNA, and HCP. Then, the influence of the MA sequence in the double flow-through process on the mAb yield and DNA and HCP removal was studied in batch mode. The best sequence of MAs was used for the final integrated double flow-through polishing process, where the MAs were directly connected via SPTFF. The flow-through of the first (anion exchange) MA was continuously diafiltrated via SPTFF to buffer conditions for the second (cation exchange) MA. In this way, DNA (< 2 ppm) and HCP (< 29 ppm) were removed with simultaneously high mAb yield. Moreover, 36 times higher throughput was obtained than with a standard process that combines a packed-bed column with CEX resin in a bind and elute mode with an AEX MA in flow-through mode.

An overview of the recent progress in Middle East Respiratory Syndrome Coronavirus (MERS-CoV) drug discovery

ABSTRACT Introduction The Middle East respiratory syndrome coronavirus (MERS-CoV) has remained a public health concern since it first emerged in 2012. Although many potential treatments for MERS-CoV have been developed and tested, none have had complete success in stopping the spread of this deadly disease. MERS-CoV replication comprises attachment, entry, fusion and replication steps. Targeting these events may lead to the creation of medications that effectively treat MERS-CoV infection. Areas covered This review updates the research on the development of inhibitors of MERS-CoV. The main topics are MERS-CoV‒related proteins and host cell proteins that are involved in viral protein activation and infection. Expert opinion Research on discovering drugs that can inhibit MERS-CoV started at a slow pace, and although efforts have steadily increased, clinical trials for new drugs specifically targeting MERS-CoV have not been extensive enough. The explosion in efforts to find new medications for the SARS-CoV-2 virus indirectly enhanced the volume of data on MERS-CoV inhibition by including MERS-CoV in drug assays. The appearance of COVID-19 completely transformed the data available on MERS-CoV inhibition. Despite the fact that new infected cases are constantly being diagnosed, there are currently no approved vaccines for or inhibitors of MERS-CoV.

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Single‐use centrifugal separator enables intensification of the clarification process in biomanufacturing of recombinant proteins

AbstractBACKGROUNDThe need to produce more biologics at lower costs and hence intensify bioprocessing has significantly improved upstream productivity (cell culture titers ≥3 g L−1 and cell densities ≥20 × 106 cells mL−1), shifting the economic and production bottleneck to downstream manufacturing. The first downstream unit operation is the clarification of the cell culture broth to remove various insoluble (such as cells and cell debris) and soluble (such as host cell DNA and proteins) contaminants, producing a feed stream suitable for further downstream purification. The high cell‐density cultures obtained in modern intensified bioprocessing have made legacy single‐use clarification trains (based primarily on depth filtration of the cell culture broth) ineffective and impractical, significantly increasing the economic and material burden of the clarification process.RESULTSWe have developed and characterized approaches that enable intensification of the clarification operation, by increasing process capacities while reducing material requirements and footprint, for single‐use manufacturing facilities. These approaches are based on primary clarification using the Alfa Laval CultureOne™ centrifuge (a continuous solid discharge disc‐stack centrifuge with a single‐use flow path) followed by secondary clarification using single‐use disposable depth filters. We show that these intensified clarification trains are suitable for processing high cell‐density cultures (6–12% packed cell volume) with significantly high yields (≥90%) and impurity clearance, along with reduced operating costs (30–60% savings).CONCLUSIONIncorporating a single‐use continuous discharge centrifuge into the clarification train enables clarification of high cell‐density cultures in single‐use manufacturing facilities and enhances process economic and environmental sustainability. © 2023 Society of Chemical Industry (SCI).

Cellular Targets of HIV-1 Protease: Just the Tip of the Iceberg?

Human immunodeficiency virus 1 (HIV-1) viral protease (PR) is one of the most studied viral enzymes and a crucial antiviral target. Despite its well-characterized role in virion maturation, an increasing body of research is starting to focus on its ability to cleave host cell proteins. Such findings are apparently in contrast with the dogma of HIV-1 PR activity being restricted to the interior of nascent virions and suggest catalytic activity within the host cell environment. Given the limited amount of PR present in the virion at the time of infection, such events mainly occur during late viral gene expression, mediated by newly synthesized Gag-Pol polyprotein precursors, rather than before proviral integration. HIV-1 PR mainly targets proteins involved in three different processes: those involved in translation, those controlling cell survival, and restriction factors responsible for innate/intrinsic antiviral responses. Indeed, by cleaving host cell translation initiation factors, HIV-1 PR can impair cap-dependent translation, thus promoting IRES-mediated translation of late viral transcripts and viral production. By targeting several apoptotic factors, it modulates cell survival, thus promoting immune evasion and viral dissemination. Additionally, HIV-1 PR counteracts restriction factors incorporated in the virion that would otherwise interfere with nascent virus vitality. Thus, HIV-1 PR appears to modulate host cell function at different times and locations during its life cycle, thereby ensuring efficient viral persistency and propagation. However, we are far from having a complete picture of PR-mediated host cell modulation, which is emerging as a field that needs further investigation.

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of≥80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

MiR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes.

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.

Depth filter material process interaction in the harvest of mammalian cells.

Upstream advances have led to increased mAb titers above 5g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems.

Bacterial infection promotes tumorigenesis of colorectal cancer via regulating CDC42 acetylation.

Increasing evidence highlights the role of bacteria in promoting tumorigenesis. The underlying mechanisms may be diverse and remain poorly understood. Here, we report that Salmonella infection leads to extensive de/acetylation changes in host cell proteins. The acetylation of mammalian cell division cycle 42 (CDC42), a member of the Rho family of GTPases involved in many crucial signaling pathways in cancer cells, is drastically reduced after bacterial infection. CDC42 is deacetylated by SIRT2 and acetylated by p300/CBP. Non-acetylated CDC42 at lysine 153 shows an impaired binding of its downstream effector PAK4 and an attenuated phosphorylation of p38 and JNK, consequently reduces cell apoptosis. The reduction in K153 acetylation also enhances the migration and invasion ability of colon cancer cells. The low level of K153 acetylation in patients with colorectal cancer (CRC) predicts a poor prognosis. Taken together, our findings suggest a new mechanism of bacterial infection-induced promotion of colorectal tumorigenesis by modulation of the CDC42-PAK axis through manipulation of CDC42 acetylation.