Pilot-scale demonstration of an end-to-end integrated and continuous biomanufacturing process.

There has been increasing momentum recently in the biopharmaceutical industry to transition from traditional batch processes to next-generation integrated and continuous biomanufacturing. This transition from batch to continuous is expected to offer several advantages which, taken together, could significantly improve access to biologics drugs for patients. Despite this recent momentum, there has not been a commercial implementation of a continuous bioprocess reported in the literature. In this study, we describe a successful pilot-scale proof-of-concept demonstration of an end-to-end integrated and continuous bioprocess for the production of a monoclonal antibody (mAb). This process incorporated all of the key unit operations found in a typical mAb production process, including the final steps of virus removal filtration, ultrafiltration, diafiltration, and formulation. The end-to-end integrated process was operated for a total of 25 days and produced a total of 4.9 kg (200 g/day or 2 g/L BRX/day) of the drug substance from a 100-L perfusion bioreactor (BRX) with acceptable product quality and minimal operator intervention. This successful proof-of-concept demonstrates that end-to-end integrated continuous bioprocessing is achievable with current technologies and represents an important step toward the realization of a commercial integrated and continuous bioprocessing process.

End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

The effects of alternating tangential flow (ATF) residence time, hydrodynamic stress, and filtration flux on high-density perfusion cell culture.

In this study, we investigated the effects of alternating tangential flow (ATF) cell separation on high-density perfusion cultures. We have developed methods to estimate theoretical residence times of cells in the ATF system and discovered that long residence times (above 75 s) correlate with decreased growth, metabolism, and productivity. We have calculated energy dissipation rates in the ATF transfer line and filter and empirically studied the impacts of increased exchange rates on cell culture, determining that increased hydrodynamic stress can lead to decreased cell size, lactate production, and specific productivity. Finally, we have conducted experiments to understand the relationship between filtration fluxes and ATF membrane fouling, finding that at fluxes above 60 L·m-2 ·day -1 , protein sieving coefficients see significant rates of decrease (greater than 1% per day). While most of these studies have been conducted with one cell line at one target viable cell density (40 million cells/ml), the general, directional knowledge arising from this study should be applicable to other conditions and programs, ultimately leading to more robust and well-designed perfusion processes.

Fully automated minicolumn chromatography.

Miniaturized chromatography columns (minicolumns) operated by automated liquid handlers are an integral part of bioprocess purification development. However, these systems can be limited in both their efficiency and accessibility. Because the minicolumn chromatography operation itself is higher throughput, the lower throughput pre- and post-operation activities become the bottleneck of the workflow. Additionally, method writing and operation of the systems while varying multiple parameters, using a design of experiments approach for example, can be error-prone and resource intensive. Here, we have developed a fully automated minicolumn chromatography system to both address these bottlenecks and improve the accessibility of these systems by allowing users to enter chromatography-relevant information through a simplified user interface. Methods have been developed to automate buffer preparation and protein solution titration leveraging modeling and integrated pH probes with feedback control. Chromatogram generation and fraction pooling has additionally been automated to improve the efficiency of post-chromatography operations. We have also demonstrated the flexibility of the system through an example run where both bind-and-elute chromatography and flowthrough chromatography experiments were performed in parallel. Additionally, all methodology and parameters to operate the system have been shared. We hope this will help interested parties improve the efficiency and accessibility of their minicolumn chromatography systems.

Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

Mammalian cells consume and metabolize various substrates from their surroundings for energy generation and biomass synthesis. Glucose and glutamine, in particular, are the primary carbon sources for proliferating cancer cells. While this combination of substrates generates static labeling patterns for use in (13)C metabolic flux analysis (MFA), the inability of single tracers to effectively label all pathways poses an obstacle for comprehensive flux determination within a given experiment. To address this issue we applied a genetic algorithm to optimize mixtures of (13)C-labeled glucose and glutamine for use in MFA. We identified tracer combinations that minimized confidence intervals in an experimentally determined flux network describing central carbon metabolism in tumor cells. Additional simulations were used to determine the robustness of the [1,2-(13)C(2)]glucose/[U-(13)C(5)]glutamine tracer combination with respect to perturbations in the network. Finally, we experimentally validated the improved performance of this tracer set relative to glucose tracers alone in a cancer cell line. This versatile method allows researchers to determine the optimal tracer combination to use for a specific metabolic network, and our findings applied to cancer cells significantly enhance the ability of MFA experiments to precisely quantify fluxes in higher organisms.

Integrated continuous production of recombinant therapeutic proteins.

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.

Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head-to-Head Comparison With Fed-Batch.

In this study, the authors compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach is applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch and ATF filtration for perfusion. Product concentration in fed-batch is 2.5 times greater than perfusion, while average productivity in perfusion is 7.5 times greater than fed-batch. PCA reveals more variability in the cell environment and metabolism during the fed-batch run. LDH measurements show that exposure of product to cell lysate is 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also has higher purity and lower half-antibody. Glycosylation is similar across both culture modes. The first perfusion harvest slice for both product types shows different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial.

An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis.

Nonstationary metabolic flux analysis (NMFA) is at present a very computationally intensive exercise, especially for large reaction networks. We applied elementary metabolite unit (EMU) theory to NMFA, dramatically reducing computational difficulty. We also introduced block decoupling, a new method that systematically and comprehensively divides EMU systems of equations into smaller subproblems to further reduce computational difficulty. These improvements led to a 5000-fold reduction in simulation times, enabling an entirely new and more complicated set of problems to be analyzed with NMFA. We simulated a series of nonstationary and stationary GC/MS measurements for a large E. coli network that was then used to estimate parameters and their associated confidence intervals. We found that fluxes could be successfully estimated using only nonstationary labeling data and external flux measurements. Addition of near-stationary and stationary time points increased the precision of most parameters. Contrary to prior reports, the precision of nonstationary estimates proved to be comparable to the precision of estimates based solely on stationary data. Finally, we applied EMU-based NMFA to experimental nonstationary measurements taken from brown adipocytes and successfully estimated fluxes and some metabolite concentrations. By using NFMA instead of traditional MFA, the experiment required only 6 h instead of 50 (the time necessary for most metabolite labeling to reach 99% of isotopic steady state).

Continuous Manufacturing of Recombinant Therapeutic Proteins: Upstream and Downstream Technologies.

Continuous biomanufacturing of recombinant therapeutic proteins offers several potential advantages over conventional batch processing, including reduced cost of goods, more flexible and responsive manufacturing facilities, and improved and consistent product quality. Although continuous approaches to various upstream and downstream unit operations have been considered and studied for decades, in recent years interest and application have accelerated. Researchers have achieved increasingly higher levels of process intensification, and have also begun to integrate different continuous unit operations into larger, holistically continuous processes. This review first discusses approaches for continuous cell culture, with a focus on perfusion-enabling cell separation technologies including gravitational, centrifugal, and acoustic settling, as well as filtration-based techniques. We follow with a review of various continuous downstream unit operations, covering categories such as clarification, chromatography, formulation, and viral inactivation and filtration. The review ends by summarizing case studies of integrated and continuous processing as reported in the literature.

13 C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures.

13 C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism of industrial cell lines. In this study, 13 C MFA was used to characterize the metabolic response of Chinese hamster ovary (CHO) cells to a novel medium variant designed to reduce ammonia production. Ammonia inhibits growth and viability of CHO cell cultures, alters glycosylation of recombinant proteins, and enhances product degradation. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel 13 C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how 13 C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism.

The business impact of an integrated continuous biomanufacturing platform for recombinant protein production.

The biotechnology industry primarily uses batch technologies to manufacture recombinant proteins. The natural evolution of other industries has shown that transitioning from batch to continuous processing can yield significant benefits. A quantitative understanding of these benefits is critical to guide the implementation of continuous processing. In this manuscript, we use process economic modeling and Monte Carlo simulations to evaluate an integrated continuous biomanufacturing (ICB) platform and conduct risk-based valuation to generate a probabilistic range of net-present values (NPVs). For a specific ten-year product portfolio, the ICB platform reduces average cost by 55% compared to conventional batch processing, considering both capital and operating expenses. The model predicts that these savings can further increase by an additional 25% in situations with higher-than-expected product demand showing the upward potential of the ICB platform. The ICB platform achieves these savings and corresponding flexibility mainly due to process intensification in both upstream and downstream unit operations. This study demonstrates the promise of continuous bioprocessing while also establishing a novel framework to quantify financial benefits of other platform process technologies.

Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

(13)C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled (13)C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-(13)C(2)]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-(13)C]glucose and [3-(13)C]glucose also outperformed the more commonly used [1-(13)C]glucose. [U-(13)C(5)]glutamine emerged as the preferred isotopic tracer for the analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of (13)C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture.

Systematic identification of conserved metabolites in GC/MS data for metabolomics and biomarker discovery.

Analysis of metabolomic profiling data from gas chromatography-mass spectrometry (GC/MS) measurements usually relies upon reference libraries of metabolite mass spectra to structurally identify and track metabolites. In general, techniques to enumerate and track unidentified metabolites are nonsystematic and require manual curation. We present a method and software implementation, freely available at http://spectconnect.mit.edu, that can systematically detect components that are conserved across samples without the need for a reference library or manual curation. We validate this approach by correctly identifying the components in a known mixture and the discriminating components in a spiked mixture. Finally, we demonstrate an application of this approach with a brief analysis of the Escherichia coli metabolome. By systematically cataloguing conserved metabolite peaks prior to data analysis methods, our approach broadens the scope of metabolomics and facilitates biomarker discovery.

Increasing the clearance of protein-bound solutes by addition of a sorbent to the dialysate.

The capacity of sorbent systems to increase solute clearances above the levels that are provided by hemodialysis has not been well defined. This study assessed the extent to which solute clearances can be increased by addition of a sorbent to the dialysate. Attention was focused on the clearance of protein-bound solutes, which are cleared poorly by conventional hemodialysis. A reservoir that contained test solutes and artificial plasma was dialyzed first with the plasma flow set at 46 +/- 3 ml/min and the dialysate flow (Q(d)) set at 42 +/- 3 ml/min using a hollow fiber kidney with mass transfer area coefficients greater than Q(d) for each of the solutes. Under these conditions, the clearance of urea (Cl(urea)) was 34 +/- 1 ml/min, whereas the clearances of the protein-bound solutes indican (Cl(ind)), p-cresol sulfate (Cl(pcs)), and p-cresol (Cl(pc)) averaged only 5 +/- 1, 4 +/- 1, and 14 +/- 1 ml/min, respectively The effect of addition of activated charcoal to the dialysate then was compared with the effect of increasing Q(d) without addition of any sorbent. Addition of charcoal increased Cl(ind), Cl(pcs), and Cl(pc) to 12 +/- 1, 9 +/- 2, and 35 +/- 4 ml/min without changing Cl(urea). Increasing Q(d) without the addition of sorbent had a similar effect on the clearance of the protein-bound solutes. Mathematical modeling predicted these changes and showed that the maximal effect of addition of a sorbent to the dialysate is equivalent to that of an unlimited increase in Q(d). These results suggest that as an adjunct to conventional hemodialysis, addition of sorbents to the dialysate could increase the clearance of protein-bound solutes without greatly altering the clearance of unbound solutes.

The clearance of protein-bound solutes by hemofiltration and hemodiafiltration.

BACKGROUND: Hemofiltration in the form of continuous venovenous hemofiltration (CVVH) is increasingly used to treat acute renal failure. Compared to hemodialysis, hemofiltration provides high clearances for large solutes but its effect on protein-bound solutes has been largely ignored. METHODS: Standard clinical systems were used to remove test solutes from a reservoir containing artificial plasma. Clearances of the protein-bound solutes phenol red (C(PR)) and indican (C(IN)) were compared to clearances of urea (C(UREA)) during hemofiltration and hemodiafiltration. A mathematical model was developed to predict clearances from values for plasma flow Q(p), dialysate flow Q(d), ultrafiltration rate Q(f), filter size and the extent of solute binding to albumin. RESULTS: When hemofiltration was performed with Q(p) 150 mL/min and Q(f) 17 mL/min, clearance values were C(PR) 1.0 +/- 0.1 mL/min; C(IN) 3.7 +/- 0.5 mL/min; and C(UREA) 14 +/- 1 mL/min. The clearance of the protein-bound solutes was approximately equal to the solute-free fraction multiplied by the ultrafiltration rate corrected for the effect of predilution. Addition of Q(d) 42 mL/min to provide HDF while Q(p) remained 150 mL/min resulted in proportional increases in the clearance of protein-bound solutes and urea. In contrast, the clearance of protein-bound solutes relative to urea increased when hemodiafiltration was performed using a larger filter and increasing Q(d) to 300 mL/min while Q(p) was lowered to 50 mL/min. The pattern of observed results was accurately predicted by mathematical modeling. CONCLUSION: In vitro measurements and mathematical modeling indicate that CVVH provides very limited clearance of protein-bound solutes. Continuous venous hemodiafiltration (CVVHDF) increases the clearance of protein-bound solutes relative to urea only when dialysate flow rate and filter size are increased above values now commonly employed.