Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10¹¹ vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10¹¹ vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.     

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

Modeling and scale-up of the unsterile scleroglucan production process with Sclerotium rolfsii ATCC 15205

An empirical model was applied to describe the growth related formation of scleroglucan in batchwise cultivation of Sclerotium rolfsii. In this case, the level of oxygen supply controls the carbon flux into glucan, biomass, and CO₂ evolution and therefore determines the yield coefficients Y_Glucan/BDM and Y_BDM/O2. It was observed that scleroglucan formation is enhanced under microaerobic conditions. However, as the empirical model and data of actual batch cultivations show, different maxima exist for product end concentration [g/l] and volumetric productivity [g/ld] depending on the total oxygen uptake during cultivation. A sufficient bulk mixing of the highly viscous culture suspension becomes particularly important during large-scale cultivations. In addition, the scleroglucan production process proved to be shear sensitive. A correlation between the attainable molecular weight of the glucan and the stirrer tip velocity in bioreactors of different sizes is presented. For all these reasons, a scale-up of this process is very complex. Large-scale cultivations under microaerobic conditions, aiming for maximum product end concentration, were slowed down by poor bulk mixing leading to a lower carbon flux into glucan formation. On the other hand, a scale-up designed for maximum volumetric productivity using high oxygen supply was successfully conducted up to a reactor volume of 1.500 l. To minimize the loss in product quality (molecular weight of the glucan) due to high stirrer tip velocities, a mixing concept was developed employing reduced agitation combined with maximum aeration to secure a sufficient axial bulk mixing in the reactor.     

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis.     

Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well.     

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.     

Immobilization of genetically engineered cells: a new strategy for higher stability.

The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.     

Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers.

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAM beta 1, which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of imparied termination of rolling-circle replication and that the impairment is due to the nature of the DNA insertion.     

A continuous process to extract plasmid DNA based on alkaline lysis

Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in <90 min. The plasmid yields are approximately 80-90 mg l(-1) culture.     

Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins. (c) 1992 John Wiley & Sons, Inc.     

Development of a robust, versatile, and scalable inoculum train for the production of a DNA vaccine

For many microbial fermentation processes, the inoculum train can have a substantial impact on process performance in terms of productivity, profitability, and process control. In general, it is understood that a well-characterized and flexible inoculum train is essential for future scale-up and implementation of the process in a pilot plant or manufacturing setting. A fermentation process utilizing E. coli DH5 for the production of plasmid DNA carrying the HIV gag gene for use as a vaccine is currently under development in our laboratory. As part of the development effort, we evaluated inoculum train schemes that incorporate one, two, or three stages. In addition, we investigated the effect of inoculum viable-cell concentrations, either thawed or actively growing, over a wide range (from 2.5 x 10(4) to 1.0 x 10(8) viable cells/mL or approximately 0.001% to 4% of final working volume). The various inoculum trains were evaluated in terms of final plasmid yield, process time, reproducibility, robustness, and feasibility at large scale. The results of these studies show that final plasmid yield remained in the desired range, despite the number of stages or inoculation viable-cell concentrations comprising the inoculum train. On the basis of these observations and because it established a large database, the first part of these investigations supports an exceptional flexibility in the design of scalable inoculum trains for this DNA vaccine process. This work also highlighted that a slightly higher level of process reproducibility, as measured by the time for the culture to reach mid-exponential growth, was observed when using actively growing versus frozen cells. It also demonstrated the existence of a viable-cell concentration threshold for the one-stage process, since we observed that inoculation of the production stage with very low amounts of viable cells from a frozen source could lead to increased process sensitivity to external factors such as variation in the quality of the raw materials used in the medium formulation. However, our analysis indicates that, despite this slight disadvantage, a one-stage inoculum train was a viable option in many situations, especially if the inoculation viable-cell concentration was kept above 4.8 x 10(6) viable cells/mL. Because it leads to a reduction in process steps and eliminates some capital investments (i.e., inoculum fermenter), when feasible a one-stage process configuration will positively impact process economics.     

Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability

Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924-926nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2-5 code for putative mobilization proteins (Mob A-D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91kbp), pRS12 (4.91kbp), pRS2 (2.996kbp), and pRS3 (2.623kbp) that bear the Spectinomycin resistance determinant (Spc(R)) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of Spc(R) constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.     

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Abstract

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (k_La) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by Mut^S P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2?kW m^?3. Specific growth rate, and then dextranase production, increased as k_La and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2?L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut⁺ strain. After a scale-up process, at constant k_La, the specific growth rate was kept at 30?L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30?L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.     

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.     

Protein fabrication automation

Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.     

Monitoring of RNA clearance in a novel plasmid DNA purification process

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.     

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.