The authors have declared that no competing interests exist.
Conceived and designed the experiments: SK AS MS-K DAO. Performed the experiments: SK AS MF ASM. Analyzed the data: SK AS MS-K DAO. Contributed reagents/materials/analysis tools: DM. Wrote the paper: AM AS. Conceptional outline and participating by adding key points to the manuscript: AM MS-K DM HVB HZ.
Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed.
The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity.
An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.
The Human Immunodeficiency Virus (HIV) continues to threaten human health. In the year 2010, around 34 million people were living with HIV as reported by the world health organization (UNAIDS World AIDS Day Report, 2011;
Neutralization assays are the common tool for evaluating neutralization profiles of HIV-infected or vaccinated individuals. A number of different assays with variable advantages and limitations are available (for summary see NeutNet Report
In order to cover the increasing demand of high quality reagents for ongoing and upcoming HIV vaccine trials, we established an automated system for production of HIV-1 Env-pseudotyped viruses. The automation includes stable cultivation of the cell line 293T/17 that is used for cell transfection and virus production. It offers the advantage of liter-scale, operator-independent and consistent virus production under GCLP guidelines. This automated HIV pseudovirus production and distribution to GCLP-compliant, international test laboratories
To cover the worldwide demand of high quality HIV-1 Env-pseudotyped viruses for antibody assessment in HIV vaccine trials, an automated production system was established. The main challenge of transferring the clearly defined steps of the manual procedure to an automated system was to combine the necessary hardware requirements with a computerized system enabling the scheduling and the connection between the single steps (
(A) Summary of the key experimental steps of the manual procedure for the production of HIV-1 Env-pseudotyped viruses. (B) Definition of the main challenges to transfer the manual process to the automated system.
(A) The complete system covered with a biosafety cabinet class II. (B) The robotic manipulator arm (RoMa) transports RoboFlasks from the incubator to the Flask Flipper at the worktable. (C) 2 RoboFlasks are fixed at the Flask Flipper and pierced by the needle of the Liquid Handling Arm (LiHa) for aspirating or dispensing liquids.
The linkage between the hardware components and the automatically performed actions is controlled by two software programs. The Freedom Evoware Plus Software controls the RoMa, the LiHa, the Flask Flipper and the transfer station. This software is also used to define and program all single steps of the individual processes. The CellGEM (Cell Growth, Expansion, Maintenance) software is of overriding importance and is responsible for scheduling the time for splitting cells, harvesting and management of cell culture parameters, disposables and reagent availability. Cell culture processes (maintenance request) or pseudovirus production (production request) were established using this software.
A major issue in the overall automation procedure was the exact enumeration of viable cells. This was solved by incorporating a Cedex Cell Counter (Roche, Innovatis), a validated automated cell counting system
(A) Cultivation of the 293T/17 cells and (B) the HIV-1 pseudovirus preparation with the adapted volumes of growth medium, PBS, Trypsin-EDTA, the incubation times and the cell numbers. (C) The performed manual steps.
The consistent quality of the cell culture within the automated system is an important criterion for the reliable supply of 293T/17 cells for HIV-1 pseudovirus production. Therefore the accurate determination of cell numbers with the Cedex Cell Counter is necessary and has been verified with ten parallel measurements of a suspension of 293T/17 cells by the Cedex Cell Counter and the Neubauer hemacytometer chamber. The results demonstrated that the cell concentrations measured by the Cedex Cell Counter never deviated more than 1.5-fold from the mean value determined with the Neubauer hemacytometer chamber thus meeting pre-defined acceptance criteria (
Illustrated are 10 measurements of a suspension of 293T/17 cells conducted by the Cedex Cell Counter and the Neubauer hemacytometer chamber. The values of Cedex counting are within 1.5-fold of the mean value of the measurements with the Neubauer hemacytometer chamber.
Before the 239T/17 cells could be used for pseudovirus preparation, stable cell growth, cell maintenance and cell viability had to be demonstrated on the automated system. For this, two independent maintenance runs were tracked over 15 passages. The number of viable cells per RoboFlask varied in request number 2120 from 10.1 to 20.9×106 and in request number 2138 from 9.5 to 22.5×106. This was well within the pre-defined limits of 6 to 30×106 cells per RoboFlask (
Shown are 2 different maintenance runs (No. 2120, No. 2138) cultivated for 15 passages with the automated system. The cell numbers range for each splitting task between the ranges of 6×106 and 30×106 cells per harvested RoboFlask.
Maintenance Run No. | Passage Number | Cell viability in (%) |
2120 | 18–24 | 91.9–96.6 |
2121 | 20–26 | 91.8–96.4 |
2127 | 34–40 | 93.6–97.5 |
2135 | 39–45 | 94.3–97.8 |
To prepare HIV-1 pseudoviruses on the automated system, the following specific scripts with defined parameters were implemented: (I) Delivery Harvesting Automation (harvest of the 293T/17 cells and the collection in a vessel), (II) Delivery Transfer (seeding of 2×106 293T/17 cells per RoboFlask), (III) Post Delivery Transfer (transfection) and (IV) Medex2 for delivery (harvest of the virus containing supernatant). In 5 different small-scale production tasks (140 ml each) the settings were established on the automated system. The quality of the automated production of small-scale pseudovirus stocks consisting of 6535.3 (tier 1B), QH0692.42 (tier 2) and PVO.4 (tier 3) all classified as HIV-1 Clade B viruses
ID50 values (µg/ml) of virus stocks determined with HIV-neutralizing test reagents | |||||
Pseudovirus | sCD4 | IgG1b12 | 2F5 | 4E10 | TriMab |
HIV-REJO4541.67 automate | 1.79 | 2.84 | 0.98 | 2.70 | 1.37 |
HIV-REJO4541.67 manual | 1.89 | 2.71 | 1.25 | 2.70 | 1.54 |
HIV-QH0692.42 automate | 3.94 | 1.13 | 6.44 | 19.33 | 1.33 |
HIV-QH0692.42 manual | 3.10 | 0.96 | 2.49 | 7.14 | 0.89 |
HIV-ZM197M.PB7 automate | 14.23 | >25 | 44.28 |
1.13 | 31.46 |
HIV-ZM197M.PB7 manual | 11.56 | 24.41 | 29.94 |
0.98 | 27.07 |
HIV-WITO4160.33 automate | 9.36 | 8.76 | 2.13 | 2.82 | 1.95 |
HIV-WITO4160.33 manual | 9.00 | 7.20 | 3.45 | 4.63 | 3.29 |
HIV-SF162.LS automate | 0.14 | 0.04 | 0.14 | 8.95 | 2.83 |
HIV-SF162.LS manual | 0.13 | 0.03 | 0.10 | 5.52 | 1.42 |
initial concentration 50 µg/µl.
In order to determine the reproducibility of the automated pseudovirus production, 5 virus stocks were prepared consecutively on the automated system using the same plasmid stocks for both backbone plasmid (pSG3Δenv) and env plasmid (SF162.LS). These preparations were compared to the preparation of a single virus stock produced with the manual procedure using the same backbone and env plasmids used in the automated production. Pseudovirus SF162.LS was chosen because it is used in neutralization experiments for HIV vaccine development due to its high neutralization sensitivity
ID50 values (µg/ml) of virus stocks determined with HIV-neutralizing test reagents | |||||
Pseudovirus | sCD4 | IgG1b12 | 2F5 | 4E10 | TriMab |
HIV-SF162.LS reference stock 1. harvest | 0.14 | 0.05 | 3.46 | 5.58 | 0.09 |
HIV-SF162.LS manual 1. harvest | 0.19 | 0.08 | 5.31 | 12.80 | 0.17 |
HIV-SF162.LS manual 2. harvest | 0.16 | 0.05 | 3.06 | 6.83 | 0.09 |
HIV-SF162.LS automate 1. harvest (batch 1) | 0.18 | 0.05 | 2.85 | 8.06 | 0.11 |
HIV-SF162.LS automate 2. harvest (batch 1) | 0.17 | 0.04 | 2.68 | 5.23 | 0.11 |
HIV-SF162.LS automate 1. harvest (batch 2) | 0.18 | 0.05 | 4.52 | 8.50 | 0.13 |
HIV-SF162.LS automate 2. harvest (batch 2) | 0.16 | 0.05 | 2.64 | 5.70 | 0.09 |
HIV-SF162.LS automate 1. harvest (batch 3) | 0.17 | 0.05 | 3.95 | 7.85 | 0.15 |
HIV-SF162.LS automate 2. harvest (batch 3) | 0.14 | 0.04 | 2.57 | 5.48 | 0.11 |
HIV-SF162.LS automate 1. harvest (batch 4) | 0.16 | 0.06 | 4.08 | 10.96 | 0.18 |
HIV-SF162.LS automate 2. harvest (batch 4) | 0.19 | 0.05 | 3.73 | 6.08 | 0.11 |
HIV-SF162.LS automate 1. harvest (batch 5) | 0.20 | 0.06 | 4.60 | 10.79 | 0.13 |
HIV-SF162.LS automate 2. harvest (batch 5) | 0.17 | 0.05 | 3.21 | 8.99 | 0.12 |
Mean of the automated productions | 0.17 | 0.05 | 3.48 | 7.76 | 0,12 |
The validation of the automated production procedure together with the associated components included the parameters accuracy, specificity, robustness and precision. Because accuracy and precision of the automated cell cultivation are linked to the reliable determination of viable cell numbers and consequently the reproducible production of HIV-1 pseudoviruses, the accuracy and precision of the pipetting volumes were verified by gravimetrical measurements using a SAG 285/01 balance from Mettler Toledo. Results of the 10 measurements of selected volumes (100 µl, 800 µl, 2500 µl and 4500 µl) met the pre-defined limits of accuracy (deviation to the target volume divided by the target volume) ≤7.0% and coefficient of variation (CV) ≤5% (data not shown). The robust and sterile maintenance conditions for cell cultivation was evaluated with 293T/17 cells maintained on the system in antibiotic-free sterile medium for 6 passages (3 weeks). Bacteria and fungi tested negative in a bioburden assay using Tryptic Soy Agar plates according to the manufacturer (heipha Dr. Müller GmbH, Eppelheim, Germany). Likewise, tests for Mycoplasma contamination by a third party testing facility (Labor Dr. Thiele, Institut für Immunologie und Genetik, Kaiserslautern, Germany) were negative, demonstrating the quality of the culture environment (data not shown). Furthermore, the system sterility was evaluated with sterile medium without antibiotics aspirated from the worktable by each of the eight channels and dispensed into 8 RoboFlasks. After 7 days at 37°C/5% CO2 and subsequent examination for microbial growth and cell growth by microscopic analysis, no contamination inside the 8 RoboFlasks was detected. Thus the sterilization procedure of the tubing and needles with 0.4% Peraclean solution within the Clean System program at the end of each process was successfully validated.
The created standard operating procedures (SOPs) directing all the operations of the automated system including cell cultivation and pseudovirus production, as well as the associated components, equipment and reagent preparations in a GCLP-compliant manner were formally audited by the Central Quality Assurance Unit (CQAU) of CAVD/CA-VIMC and externally audited by PPD Laboratory Services and Sailstad & Associates. With this, the established automated HIV-1 pseudovirus preparation system is ready to produce high quality reagents for worldwide vaccine trials.
Here we present the results of the transfer of a manual production procedure for HIV-1 pseudoviruses to an automated system in a GCLP-compliant manner. This automated system consists of a modified Tecan-based Cellerity system and performs the complete production process with several dependent steps under computer control. It allows the accurate production of liter-scale pseudovirus stocks with concordant quality. In addition, the reproducibility and robustness of the production procedure was demonstrated by the generation of 5 batches of the Clade B pseudovirus HIV-SF162.LS using the same plasmid stocks. Besides the independency from external influences, the advantage of the automated system is the day and night operation that allows the stable and scalable supply of the producer cell line 293T/17. Other beneficial outcomes of this automated method are the robust and sterile production conditions with minimal manual interactions such as the provision of the transfection mix and the weekly maintenance. The automatic monitoring of the complete production process via the generated maintenance reports facilitates the traceability of all individual steps. In addition, an email notification system has been established to inform the personnel about state change, depletion of resources and potential errors so that necessary steps can be initiated immediately. Furthermore, the temperatures of the refrigerator, worktables and reagents troughs are tracked automatically in order to guarantee the stability of the production environment. All electronic information is saved on a server for long-term storage and retrieval if need occurs. Given this functional prototype automation, procedure modifications to produce other reagents including replication competent viruses like those with incorporated marker genes
Automated cell cultivation plays an increasing role in numerous fields of science providing the advantage of removing process variability due to operator dependency and reducing occupation of personnel with repetitive routine tasks
For the automation described here, GCLP standards
In conclusion, we have assembled a complete, GCLP-validated, automated unit for reliable cell cultivation, transfection and HIV pseudovirus production of high quality. Its large production scale combined with its high reproducibility will facilitate reagent production for ongoing and upcoming HIV vaccine trials. As the overall set-up of this prototype system is very flexible, other applications involving cell cultivation, transfection and harvesting of supernatants may easily be implemented. Thus this system holds great promise for future production of biological reagents of diverse applications.
The TZM-bl cell line was obtained through the NIH AIDS Research and Reference Reagent Program (ARRRP, catalog no. 8129), as contributed by J. Kappes and X. Wu
The manual HIV pseudovirus production procedure in T-75 culture flask was described previously
The 50% tissue culture infectious dose (TCID) of a single thawed aliquot of each batch of pseudovirus was determined in TZM-bl cells as described elsewhere
Neutralizing antibodies were measured as reductions in Luciferase reporter gene expression after a single round of virus infection of TZM-bl cells as described previously
Validation parameters evaluated in this study included accuracy, specificity, robustness and precision. The accuracy of the cell density was assayed using standard reference beads (Roche) with a defined concentration (10.06×105/ml) and the suspension of the 293T/17 cells seeded with the density of 2×106 in each of three T-75 cell culture flasks for counting after 24 h, 48 h, 72 h and 96 h. The reference beads and the cells of each flask were counted in parallel via the Neubauer hemacytometer chamber and the Cedex Cell Counter. On the basis of these results (data not shown), acceptance criteria were defined: the values measured by the Cedex Cell Counter must be within 1.5-fold compared to the Neubauer chamber.
The limit to test the accuracy and precision of the automatically produced HIV-1 Env-pseudotyped viruses was set by preparing five different virus stocks (QH0692.42, 6535.3, PVO.4, SF162.LS and MN.3) in small-scale (140 ml of each) and evaluating them with the bridging test in the form of two neutralization assays performed in parallel. The test was considered positive if the neutralization titers for at least 80% of the assayed reagents (sCD4, IgG1b12, 2F5, 4E10 and TriMab) agree within 3-fold between the data of the automatically produced pseudovirus and the manual prepared reference stock, corresponding the outcomes of the bridging tests of the small scale automated prepared pseudoviruses (Table S2). Subsequent to these results the pass criterion was defined: the neutralization titers of four of the five test reagents must be within 3-fold.
Robustness and specificity of the automated cell cultivation of the 293T/17 cells was implemented with respect to the number of harvested cells after 84 h of incubation on the automate and the viability. On the basis of the result of the maintenance run transferring the 293T/17 cells within the system over 13 passages, the number of harvested 293T/17 cells per RoboFlasks must range between 6×106 and 30×106 to establish a stable cell line (data not shown).
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We thank Ciro Miniaci, Daniel Langendörfer and Horst Noecker from TECAN for their support in the assembly of the automated system and during the validation phase. Further we thank Kelli M. Greene and Hongmei Gao for their contributions in project management, Ralf Oschwald and Ursula Spychala for their assistance regarding the quality assurance as well as Anja Germann, Markus Michel, Frank Obergrießer, Young-Joo Oh, Jochen Schmidt and Uwe Schön for the technical support that made the realization of this project possible.