RESUMO
Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.
Assuntos
Proteômica , Proteômica/métodos , Ensaios de Triagem em Larga Escala , Animais , Cromatografia Líquida/métodos , Cricetulus , Células CHO , Humanos , Espectrometria de MassasRESUMO
Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.
Assuntos
Anticorpos Monoclonais , Filtração , Cricetinae , Animais , Cricetulus , Células CHO , Perfusão , Filtração/métodos , Reatores Biológicos , Membranas ArtificiaisRESUMO
Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.
Assuntos
Ativação Plaquetária , Adesividade Plaquetária , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/metabolismo , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Adulto , Animais , Sítios de Ligação , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/genética , Adesividade Plaquetária/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/farmacologia , Transfecção , Fator de von Willebrand/genética , Fator de von Willebrand/farmacologiaRESUMO
Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.
RESUMO
The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders.
Assuntos
Cromossomos Humanos Y/metabolismo , Epigênese Genética , Eucromatina/metabolismo , Sítios de Ligação , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Cromossomos Humanos Y/química , Eucromatina/química , Expressão Gênica , Genes sry , Loci Gênicos , Histonas/metabolismo , Humanos , Masculino , Proteínas Repressoras/metabolismo , Sequências de Repetição em TandemRESUMO
Biologics encompasses a wide variety of therapeutics including monoclonal antibodies, fusion proteins, and enzymes, among others. The biologics market is growing at a rapid pace and different manufacturing processes, including continuous manufacturing processes, are being increasingly adopted. There is a strong drive to assess the sustainability of such processes. Here, we calculated the process mass intensity (PMI) of a continuous manufacturing process and compared it to the PMI of batch processes for monoclonal antibodies (mAbs). Results show that the PMI of continuous manufacturing process is comparable to that of batch processes. Sensitivity analysis was performed to assess the impact of different process strategies on the material usage efficiency of continuous processes. Although PMI is a useful benchmarking metric of sustainability, it does not account for factors such as energy consumption which is a key driver of sustainability for biologics manufacturing. Comparison of a higher PMI continuous process with a lower PMI batch process operating at the same bioreactor scale shows that since the productivity (in g of drug substance, DS) per unit time is multifold higher for the continuous process, the overall energy consumption per unit of DS produced might be lower leading to a more environmentally sustainable process. This study highlights some of these key aspects that would require additional metrics and models to be developed to assess the overall sustainability of biologics processes.
Assuntos
Produtos Biológicos , Reatores Biológicos , Anticorpos MonoclonaisRESUMO
The rapid growth of biologics as the preferred modality in several therapeutic areas has led to changes in the environmental profile of pharmaceutical manufacturing for some companies. The increased use of single use technologies (SUT) in biologics manufacturing has been accompanied by a greater public awareness of plastics waste, but the full life cycle environmental impacts of SUT have had limited study. Therefore, a segment of American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable member companies undertook a streamlined cradle-to-gate life cycle assessment on a biological bulk drug substance (BDS) manufacturing process utilizing SUT at the 2000 L scale. The goal of this study was to highlight where pharmaceutical companies, and biologics producers in particular, can reduce the environmental impact of their drug substance manufacturing. The results have shown that the largest contribution to the life cycle environmental impact for SUT was found to be the electricity used to operate the plant. Interestingly, across all impact categories, the contribution to the environmental footprint from end-of-life due to the use of plastic SUT was extremely small. Although not quantified in this study, these findings and others suggest operational changes that increase process efficiency and decrease time in plant are among the best strategies for reducing the life cycle environmental impact of biologics manufacturing.
Assuntos
Produtos Biológicos , Animais , Meio Ambiente , Estágios do Ciclo de VidaRESUMO
Intensified perfusion processes are an integral part of continuous manufacturing for biopharmaceuticals which enable agile operations and significant reduction in cost of goods. However, they require large volumes of media to support robust cell growth and maintain high productivity, posing substantial challenges to operations, logistics, and process sustainability. This study explores a novel strategy for reprocessing and reusing permeate from perfusion cultures for mAb production. The concept was initially evaluated by recycling permeate, Protein A flow-through (ProA FT) and CEX processed ProA FT in deep-well plate mock perfusion and ambr® 250 perfusion formats. Further processing of ProA FT through a cation exchange depth filter before recycling reduced process impurities such as host cell proteins (HCPs) and DNA. However, a direct replacement of fresh media with spent media reduces nutrient depth which results in a concomitant reduction in productivity. In ambr® 250 bioreactors, recycling of ProA FT at 25%-50% replacement rates (defined as the fraction of recycled material in media) resulted in a 13%-30% reduction in cumulative productivity while maintaining product quality. To mitigate this, we used media concentrates which allowed independent modulation of media depth by replacing a portion of diluent WFI with recycled material. Results from deep-well mock perfusion studies demonstrated that comparable or higher productivities relative to control can be achieved with this approach. Taken together, our study demonstrates the feasibility of recycling permeate in perfusion cultures. Process mass intensity (PMI) calculations reveal that this approach can meaningfully improve material efficiency by reducing water consumption, thereby enhancing overall bioprocess sustainability.
Assuntos
Produtos Biológicos , Animais , Reatores Biológicos , Proliferação de Células , Cricetinae , Cricetulus , Perfusão , Proteína Estafilocócica ARESUMO
Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.
Assuntos
Anticorpos Monoclonais , Polissacarídeos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , GlicosilaçãoRESUMO
Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein-protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfide to covalently crosslink the subunits of a complex, so that the disulfide-trapped complex can be displayed on the yeast surface for detection and analysis. Transient protein complexes are difficult to display on the yeast surface, since they may dissociate before they can be detected due to a long induction period in yeast. To this end, we show that three different quaternary structures with the subunit dissociation constant K(d) approximately 0.5-20 microM, the antibody variable domain (Fv), the IL-8 dimer, and the p53-MDM2 complex, cannot be displayed on the yeast surface as a noncovalent complex. However, when we introduce an interchain disulfide between the subunits, all three systems are efficiently displayed on the yeast surface, showing that disulfide trapping can help display protein complexes that cannot be displayed otherwise. We also demonstrate that a disulfide forms only between the subunits that interact specifically, the displayed complexes exhibit functional characteristics that are expected of wt proteins, the mutations that decrease the affinity of subunit interaction also reduce the display efficiency, and most of the disulfide stabilized complexes are formed within the secretory pathway during export to the surface. Disulfide crosslinking is therefore a convenient way to study weak protein association in the context of yeast display.
Assuntos
Dissulfetos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cinética , Proteínas de Membrana/química , Modelos Moleculares , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Process mass intensity (PMI) is a benchmarking metric to evaluate the efficiency of a manufacturing process, which is indicative of the environmental impact of the process. Although this metric is commonly applied for small molecule manufacturing processes, it is less commonly applied to biologics. In this study, an Excel based tool developed by the ACS GCI Pharmaceutical Roundtable was used to calculate PMI of different manufacturing processes for a monoclonal antibody (mAb). For the upstream process, three different versions were compared: fed-batch, fed-batch with N-1 perfusion, and perfusion in the N-stage bioreactor. For each upstream process version, an appropriate downstream operational mode was evaluated from the following: a column chromatography process utilizing Protein A and anion exchange (AEX) resin, a Protein A column and an AEX membrane, and a three-column periodic counter-current (3C PCC) chromatography process for Protein A and an AEX membrane. The impact of these different process variations on PMI was evaluated. Of all the process inputs, water contributes about 92-94% of the overall PMI. Additionally, the upstream processes and the chromatography steps account for 32-47 and 34-54% of the overall PMI, respectively. Sensitivity analysis was performed to identify opportunities for further reducing PMI. These data indicate that a semicontinuous manufacturing process (perfusion, 3C PCC, and AEX membrane) is the most efficient process, resulting in a 23% reduction of PMI when compared with the fed batch and two-column chromatography process. Together, PMI can be used to guide the development of efficient and environmentally sustainable mAb manufacturing processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1566-1573, 2018.
Assuntos
Cromatografia/métodos , Proteína Estafilocócica A/química , Anticorpos Monoclonais/química , Reatores BiológicosRESUMO
BACKGROUND: Von Willebrand Factor (VWF) A1-domain binding to platelet receptor GpIbα is an important fluid-shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N-terminus of the A1-domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D'D3-domain, A1-domain N-terminal flanking peptide (NFP), and O-glycans on this peptide. METHODS AND RESULTS: Full-length dimeric VWF (ΔPro-VWF), dimeric VWF lacking the D'D3 domain (ΔD'D3-VWF), and ΔD'D3-VWF variants lacking either the NFP (ΔD'D3NFP(â)-VWF) or just O-glycans on this peptide (ΔD'D3OG(â)-VWF) were expressed. Monomeric VWF-A1 and D'D3-A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro-VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50- to 100-fold higher than other proteins lacking the D'D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on-rate of D'D3-A1 binding to immobilized GpIbα (kon=1.8±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.7 µmol/L) was reduced compared with the single VWF-A1 domain (kon=5.1±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.2 µmol/L). Thus, VWF-D'D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1-GpIbα binding. Here, in ELISA, the number of apparent A1-domain sites available for binding GpIbα on ΔPro-VWF was ≈50% that of the ΔD'D3-VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro-VWF<ΔD'D3-VWF<ΔD'D3NFP(â)-VWF<ΔD'D3OG(â)-VWF. CONCLUSIONS: Whereas VWF-D'D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D'D3-domain and N-terminal peptide regulate platelet translocation and thrombus formation.