Development of cancer biomarker heat shock protein 90α certified reference material using two different isotope dilution mass spectrometry techniques.

Heat shock protein 90α (HSP90α) has been regarded as an important indicator for judging tumor metastasis and prognosis due to its significant upregulation in various tumors. Therefore, the accurate quantification of HSP90α is of great significance for clinical diagnosis and therapy of cancers. However, the lack of HSP90α certified reference material (CRM) leads to the accuracy and consistency of quantification methods not being effectively evaluated. Besides, quantitative results without traceability make comparisons between different studies difficult. In this study, an HSP90α solution CRM was developed from the recombinant protein raw material. The recombinant protein is a dimer, and the purity of the CRM candidate reached 96.71%. Both amino acid analysis-isotope dilution mass spectrometry (AAA-IDMS) and unique peptide analysis-isotope dilution mass spectrometry (UPA-IDMS) were performed to measure the content of HSP90α in the solution CRM candidate, and the certified value was assessed to be 66.2 ± 8.8 µg/g. Good homogeneity of the CRM was identified, and the stability examination suggested that the CRM was stable for at least 4 months at - 80 °C and for 7 days at 4 °C. With traceability to SI unit (kg), this CRM has potential to help establish a metrological traceability chain for quantification of HSP90α, which will make the quantification results standardized and comparable regardless of the quantitative methods.

Modulable 3D-printed plantibody-laden platform enabling microscale affinity extraction and ratiometric front-face fluorescence detection of microcystin-LR in marine waters.

A 3D-printed stereolithographic platform for selective biorecognition is designed to enable convective microscale affinity extraction of microcystin-LR (MC-LR) followed by direct solid-phase optosensing exploiting ratiometric front-face fluorescence spectroscopy. For this purpose, a recombinant monoclonal plantibody (recAb) is covalently attached to a 3D-printed structure for sorptive immunoextraction, whereupon the free and unbound primary amino moieties of the recAb are derivatized with a fluorescent probe. The fluorophore-recAb-MC-LR laden device is then accommodated in the cuvette holder of a conventional fluorometer without any instrumental modification for the recording of the solid-phase fluorescence emission. Using Rodbard's four-parameter sigmoidal function, the 3D-printed bioselective platform features a limit of detection (LOD) of 28 ng L-1 using a sample volume of 500 mL, device-to-device reproducibility down to 12%, and relative recoveries ranging from 91 to 100% in marine waters. Printed prototypes are affordable, just 0.4 € per print and ≤ 10 € per device containing recAb. One of the main assets of the miniaturized immunoextraction device is that it performs comparably well in terms of analytical figures of merit with costly mass spectrometric-based analytical methodologies, such as HPLC-MS/MS. The device is readily applicable to high-matrix samples, such as seawater, as opposed to previous biosensing platforms, just applied to freshwater systems.

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association.

von Willebrand factor (VWF) is an ultralong concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to 0 at the downstream free end. Using single-molecule fluorescence microscopy, we directly visualized the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules. We showed that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ∼10 pN, and plateaus above ∼25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered primary VWF molecules can recruit more than their own mass of secondary VWF molecules from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may occur because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. Glycoprotein Ibα (GPIbα) binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.

Double-Labeling Method for Visualization and Quantification of Membrane-Associated Proteins in Lactococcus lactis.

Lactococcus lactis displaying recombinant proteins on its surface can be used as a potential drug delivery vector in prophylactic medication and therapeutic treatments for many diseases. These applications enable live-cell mucosal and oral administration, providing painless, needle-free solutions and triggering robust immune response at the site of pathogen entry. Immunization requires quantitative control of antigens and, ideally, a complete understanding of the bacterial processing mechanism applied to the target proteins. In this study, we propose a double-labeling method based on a conjugated dye specific for a recombinantly introduced polyhistidine tag (to visualize surface-exposed proteins) and a membrane-permeable dye specific for a tetra-cysteine tag (to visualize cytoplasmic proteins), combined with a method to block the labeling of surface-exposed tetra-cysteine tags, to clearly obtain location-specific signals of the two dyes. This allows simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm. Using this method, we were able to detect full-length peptide chains for the model proteins HtrA and BmpA in L. lactis, which are associated with the cell membrane by two different attachment modes, and thus confirm that membrane-associated proteins in L. lactis are secreted using the Sec-dependent post-translational pathway. We were able to quantitatively follow cytoplasmic protein production and accumulation and subsequent export and surface attachment, which provides a convenient tool for monitoring these processes for cell surface display applications.

A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid.

This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Null-producing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformance-reversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) "fingerprints." Using these fingerprints, an inner product vector-based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null-producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, "self-orthogonality" was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor-intensive HCP tracking has important implications for efficient process development and resin/operating condition selection for both monoclonal antibody (mAb) polishing steps and non-mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next-generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems.

Combined gene and environmental engineering offers a synergetic strategy to enhance r-protein production in Chinese hamster ovary cells.

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and qP (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes.

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA-binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 µM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl ß-d-galactoside (pNPGal, Kd = 54 ± 6 µM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis.

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

Characterization and monitoring of charge variants of a recombinant monoclonal antibody using microfluidic capillary electrophoresis-mass spectrometry.

Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.

High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery.

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

Simple, rapidly electroassembled thiolated PEG-based sensor interfaces enable rapid interrogation of antibody titer and glycosylation.

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal ß-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings.

Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth.

Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early-stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate-protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L-1 with TSFS|| . Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at-line measurements, and the method had an LOD of 0.18 g L-1 and high accuracy with a predictive error of <9%.

Hydrocyclones as cell retention devices for an N-1 perfusion bioreactor linked to a continuous-flow stirred tank production bioreactor.

A continuous Chinese hamster ovary (CHO) cell culture process comprised of a highly proliferative N-1 perfusion bioreactor utilizing a hydrocyclone as a cell retention device linked to a production continuous-flow stirred tank reactor (CSTR) is presented. The overflow stream from the hydrocyclone, which is only partially depleted of cells, provides a continuous source of high viability cells from the N-1 perfusion bioreactor to the 5-20 times larger CSTR. Under steady-state conditions, this linked-bioreactor system achieved a peak volumetric productivity of 0.96 g/L/day, twofold higher than the optimized fed-batch process. The linked bioreactor system using a hydrocyclone was also shown to be 1.8-3.1 times more productive than a dual, cascading CSTR system without cell retention.

A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.