Biotechnology Progress最新文献

Biopharmaceutical manufacture is transitioning from batch to integrated and continuous biomanufacturing (ICB). The common framework for most ICB, potentially enables a global biomanufacturing ecosystem utilizing modular and multi-function manufacturing equipment. Integrating unit operation hardware and software from multiple suppliers, complex supply chains enabled by multiple customized single-use flow paths, and large volume buffer production/storage make this ICB vision difficult to achieve with commercially available manufacturing equipment. Thus, we developed SymphonX™, a downstream processing skid with advanced buffer management capabilities, a single disposable generic flow path design that provides plug-and-play flexibility across all downstream unit operations and a single interface to reduce operational risk. Designed for multi-product and multi-process cGMP facilities, SymphonX™ can perform stand-alone batch processing or ICB. This study utilized an Apollo™ X CHO-DG44 mAb-expressing cell line in a steady-state perfusion bioreactor, harvesting product continuously with a cell retention device and connected SymphonX™ purification skids. The downstream process used the same chemistry (resins, buffer composition, membrane composition) as our historical batch processing platform, with SymphonX™ in-line conditioning and buffer concentrates. We used surge vessels between unit operations, single-column chromatography (protein A, cation and anion exchange) and two-tank batch virus inactivation. After the first polishing step (cation exchange), we continuously pooled product for 6 days. These 6 day pools were processed in batch-mode from anion exchange to bulk drug substance. This manufacturing scale proof-of-concept ICB produced 0.54 kg/day of drug substance with consistent product quality attributes and demonstrated successful bioburden control for unit-operations undergoing continuous operation.

Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High-throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench-scale glass fermentors or small-scale steam-in-place (SIP) fermentors. However, such scale-down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small-scale models. In the current study, we developed a representative scale-down model of a Biostat® D-DCU 30 L pDNA fermentation process in Ambr® 250 HT fermentors using three cell lines producing three different constructs. The Ambr scale-down model provided comparable process performance and pDNA quality as the 30 L SIP fermentation process. In addition, we demonstrated the predictive value of the Ambr model by two-way qualification, first by accurately reproducing the prior trends observed in a 30 L process, followed by predicting new process trends that were then successfully reproduced in the 30 L process. The representative and predictive scale-down Ambr model developed in this study would enable a faster and more efficient approach to strain/clone/host-cell screening, pDNA process development and characterization studies, process scale-up studies, and manufacturing support.

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm² and 10 CFU/mL present in both surface and water samples, respectively.

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre-transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of “high-risk” HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody-producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE-15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE-15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE-15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >10⁶ cells/mL. Supplementation of the cultures with SE-15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess.

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process. With the aim of finding an easy-to-use alternative to FACS, we identified a flow cytometry-based microfluidic cell dispenser, which presents a single cell sorting solution for biopharmaceutical CLD. The microfluidic cell dispenser is small, budget-friendly, easy-to-use, requires lower-cost consumables, permits flow cytometry-enabled multiparametric target cell enrichment and offers fast and gentle single cell dispensing into multiwell plates. Following comprehensive evaluation, we found that single cell deposition by the microfluidic cell dispenser resulted in >99% probability of monoclonality for production cell lines. Moreover, the clonally derived producer cell lines generated from the microfluidic cell dispenser demonstrated comparable or improved growth profiles and production capability compared to the FACS derived cell lines. Taken together, microfluidic cell dispensing can serve as a cost-effective, efficient and convenient alternative to FACS, simplifying the biopharmaceutical CLD platform with significant reductions in both scientist time and running costs.

In this investigation, low molecular weight polyethyleneimine (LMW PEI; 1.8 kDa branched PEI) was conjugated to phathalated dextrin. The aim of this chemical modification was to decorate PEI molecules with a hydrophilic layer to improve its biophysical properties while the phthalic moiety may improve the hydrophilic-hydrophobic balance of the final structure. The polymers were prepared at various conjugation degrees ranging from 6.5% to 16.5% and characterized in terms of biophysical characteristics as well as their gene transfer ability and cell-induced toxicity. The results showed that dextrin-phthalated-PEI (DPHPEI) polymer was able to form nanoparticles with the size range of around 118–170 nm, with the zeta potential of 6.2–9.5 mV. DPHPEI polymers could increase the level of desired protein expression in the cells by up to three folds compared with unmodified LMW PEI while the cell viability of the modified polymers was around 80%. The result of this study shows a promising approach to improve the transfection efficiency of LMW PEI while maintaining its low toxic effects.

The move to integrated continuous bioprocessing (ICB), while providing a means for process intensification, can put added strain on process analytics when conventional methods are used. For instance, traditional microbial methods provide minimal value to ICB processes given that the time required for data to become available is much longer than a typical full cycle of the manufacturing process. Although rapid microbial detection has been in discussion for over 30 years, it is still not routinely deployed in commercial biopharmaceutical manufacturing. One contributing factor is the ability to integrate this technology into a process control strategy and existing quality systems. An understanding of the capability of microbial detection technology available today can be leveraged to implement a control strategy for bioburden monitoring in real time for process intermediates. One key tenet of this proposed control strategy is the use of a “two-tiered approach” wherein a fast (but possibly less sensitive) test is used to monitor the process and trigger further action for a second, longer duration test which is used to confirm and quantify the presence of bioburden and identify the organism. This approach, presented here alongside several case studies for microbial monitoring, can have broader application for other process analytical technologies where fit for purpose methods could be employed to establish process control alongside real time continuous processes.