RESUMO
Therapeutic monoclonal antibodies (mAbs) are highly heterogeneous as a result of posttranslational modifications (PTMs) during bioprocessing and storage. The modifications that impact mAb product quality are regarded as critical quality attributes and require monitoring. The conventional LC-mass spectrometer (MS) method used for product quality monitoring may require protein A purification prior to analysis. In this paper, we present a high-throughput microchip electrophoresis (<4 min) in-line with MS (MCE-MS) that enables baseline separation and characterization of Fc, Fd', and light chain (LC) domains of IdeS-treated mAb sample directly from bioreactor. The NISTmAb was used to optimize the MCE separation and to assess its capability of multiple attribute monitoring. The MCE-MS can uniquely separate and characterize deamidated species at domain level compared to LC-MS method. Two case studies were followed to demonstrate the method capability of monitoring product quality of mAb samples from stability studies or directly from bioreactors.
Assuntos
Anticorpos Monoclonais , Eletroforese em Microchip , Anticorpos Monoclonais/análise , Espectrometria de Massas/métodos , Processamento de Proteína Pós-TraducionalRESUMO
An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.
Assuntos
Produtos Biológicos , Eletroforese Capilar , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Laboratórios , Isoformas de ProteínasRESUMO
Residence time distribution modeling of integrated perfusion to capture process can elucidate the impact of product quality excursions and filter fouling on monoclonal antibody production. In this case study, a glycosylation inhibitor and fluorescently labeled antibody are applied to the continuous process to study protein quality modulation, perfusion filter fouling, and unit operation hold times. The unit operations were modeled as continuous-stirred tank reactors and the residence time distribution of a small molecule glycan inhibitor and impact on glycosylation were characterized. A fluorescently labeled antibody was applied as a tracer molecule and confirmed the impact of packed cell volume and filter fouling. This study demonstrates how a biologics continuous process can be modeled and characterized through residence time distribution to ensure a robust, well-understood process.
Assuntos
Anticorpos Monoclonais/biossíntese , Reatores Biológicos , Animais , Células CHO , Cricetulus , Glicosilação , PerfusãoRESUMO
UNLABELLED: The dimorphic alphaproteobacterium Prosthecomicrobium hirschii has both short-stalked and long-stalked morphotypes. Notably, these morphologies do not arise from transitions in a cell cycle. Instead, the maternal cell morphology is typically reproduced in daughter cells, which results in microcolonies of a single cell type. In this work, we further characterized the short-stalked cells and found that these cells have a Caulobacter-like life cycle in which cell division leads to the generation of two morphologically distinct daughter cells. Using a microfluidic device and total internal reflection fluorescence (TIRF) microscopy, we observed that motile short-stalked cells attach to a surface by means of a polar adhesin. Cells attached at their poles elongate and ultimately release motile daughter cells. Robust biofilm growth occurs in the microfluidic device, enabling the collection of synchronous motile cells and downstream analysis of cell growth and attachment. Analysis of a draft P. hirschii genome sequence indicates the presence of CtrA-dependent cell cycle regulation. This characterization of P. hirschii will enable future studies on the mechanisms underlying complex morphologies and polymorphic cell cycles. IMPORTANCE: Bacterial cell shape plays a critical role in regulating important behaviors, such as attachment to surfaces, motility, predation, and cellular differentiation; however, most studies on these behaviors focus on bacteria with relatively simple morphologies, such as rods and spheres. Notably, complex morphologies abound throughout the bacteria, with striking examples, such as P. hirschii, found within the stalked Alphaproteobacteria. P. hirschii is an outstanding candidate for studies of complex morphology generation and polymorphic cell cycles. Here, the cell cycle and genome of P. hirschii are characterized. This work sets the stage for future studies of the impact of complex cell shapes on bacterial behaviors.
Assuntos
Alphaproteobacteria/citologia , Alphaproteobacteria/fisiologia , Ciclo Celular/fisiologia , Técnicas Bacteriológicas , Biofilmes/crescimento & desenvolvimentoRESUMO
We describe a microfluidic device with an integrated nanochannel array to trap individual bacteria and monitor growth and reproduction of lineages over multiple generations. Our poly(dimethylsiloxane) device comprises a pneumatically actuated nanochannel array that includes 1280 channels with widths from 600 to 1000 nm to actively trap diverse bacteria. Integrated pumps and valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading and nutrient flow, permitting chemostatic growth for extended periods of time (typically 12 to 20 h). Nanochannels confine bacterial growth to a single dimension, facilitating high-resolution, time-lapse imaging and tracking of individual cells. We use the device to monitor the growth of single bacterial cells that undergo symmetric (Bacillus subtilis) and asymmetric (Caulobacter crescentus) division and reconstruct their lineages to correlate growth measurements through time and among related cells. Furthermore, we monitor the motility state of single B. subtilis cells across multiple generations by the expression of a fluorescent reporter protein and observe that the state of the epigenetic switch is correlated over five generations. Our device allows imaging of cellular lineages with high spatiotemporal resolution to facilitate the analysis of biological processes spanning multiple generations.
Assuntos
Bacillus subtilis/isolamento & purificação , Caulobacter crescentus/isolamento & purificação , Técnicas Analíticas Microfluídicas , Nanotecnologia , Bacillus subtilis/citologia , Caulobacter crescentus/citologia , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentaçãoRESUMO
We report the development of an automated microfluidic "baby machine" to synchronize the bacterium Caulobacter crescentus on-chip and to move the synchronized populations downstream for analysis. The microfluidic device is fabricated from three layers of poly(dimethylsiloxane) and has integrated pumps and valves to control the movement of cells and media. This synchronization method decreases incubation time and media consumption and improves synchrony quality compared to the conventional plate-release technique. Synchronized populations are collected from the device at intervals as short as 10 min and at any time over four days. Flow cytometry and fluorescence cell tracking are used to determine synchrony quality, and cell populations synchronized in minimal growth medium with 0.2% glucose (M2G) and peptone yeast extract (PYE) medium contain >70% and >80% swarmer cells, respectively. Our on-chip method overcomes limitations with conventional physical separation methods that consume large volumes of media, require manual manipulations, have lengthy incubation times, are limited to one collection, and lack precise temporal control of collection times.
Assuntos
Caulobacter crescentus/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Biofilmes/crescimento & desenvolvimento , Caulobacter crescentus/fisiologia , Meios de Cultura/metabolismo , Desenho de Equipamento , Microscopia de FluorescênciaRESUMO
Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization. This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques. The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.
Assuntos
Anticorpos Monoclonais/análise , Produtos Biológicos/análise , Eletroforese/métodos , Fracionamento por Campo e Fluxo/métodos , Substituição de Aminoácidos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Produtos Biológicos/química , Química Farmacêutica , Glicosilação , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Current lab-on-a-chip (LoC) devices are assay-specific and are custom-built for each single experiment. Performing an experiment requires scientists or engineers to go through the time-consuming process of designing, fabricating, and testing a chip before conducting the actual experiment. This prolonged cycle can take months to complete, increasing effort and cost and reducing productivity. Similarly, minor modifications to an assay protocol re-incur the overheads of the design cycle. In this paper, we develop a multi-purpose, software-programmableLab-on-a-Chip (SPLoC), where the user simply writes or downloads a program for each experiment. We describe the components necessary to realize the SPLoC, which include a high-level programming language, an abstract instruction set, a runtime and control system, and a microfluidic device. We describe two key features of our high-level language compiler, and describe a novel variable-volume variable-ratio mixer. Finally, we demonstrate our SPLoC on four diverse, real-world assays.