Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.     

A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy

We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing therapeutic transgenes for clinical trials. HEK 293 cells – which support the propagation of E1 deficient adenovirus – were first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in HEK 293 as well as in other cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.     

293 cell cycle synchronisation adenovirus vector production

As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 10⁶ cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 10⁶ cell/mL at infection time has usually been utilized due to the so called “cell density effect”. In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6‐fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3‐fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

Approaches for recombinant human factor IX production in serum-free suspension cultures

Objective

To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX).

Results

After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml.

Conclusion

The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.

     

重组人神经生长因子rh-β-NGF真核表达载体的构建及其在HEK293细胞中的表达

为大量制备β-NGF,构建了一种稳定、高效表达重组人神经生长因子(Recombinant human nerve growth factor,rh-β-NGF)的真核表达载体及含该重组载体的HEK293细胞株。首先,构建重组质粒p CMV-β-NGF-IRES-dhfr并转染至HEK293细胞系,用MTX加压筛选和有限稀释法进行选择,获得高效表达rh-β-NGF的单克隆重组细胞株;随后逐步降低血清培养,最终使细胞株完全适应无血清培养基并稳定表达rh-β-NGF;SDS-PAGE分析该表达产物,可见相对分子质量约13 k Da的条带,纯度大于50%,经质谱法测定得到其肽图谱与理论序列完全匹配,接着利用离子交换层析和分子筛层析纯化rh-β-NGF;最后进行重组细胞株表达效率和表达稳定性检测,表明重组细胞株可稳定、高效表达rh-β-NGF,其分泌效率大于20 pg/(cell?d),并能诱导PC12细胞的分化,具有良好的生物学活性。     

高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

由于各种疾病在全球范围内的肆虐,国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加,而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量,新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合,有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型,使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示,在细胞生长阶段使用370 mOsm的高渗透压培养基,在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中,Adv的产量达到3.2×10¹⁰ IFU/mL,是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv,同时揭示了高渗胁迫工艺增产Adv的可能原因,为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10⁷ cells cm⁻³-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.     

Viral vectors for the treatment of alcoholism: Use of metabolic flux analysis for cell cultivation and vector production

The HEK293 cell line has been used for the production of adenovirus vectors to be used in the potential treatment of alcoholism using a gene therapy strategy. Culture optimization and scale-up has been achieved by first adapting the cells to serum-free media and secondly by growing them in suspension. Adenovirus production after infection was increased, resulting in higher specific glucose consumption and lactate accumulation rates compared to the growth phase. We applied media design tools and Metabolic Flux Analysis (MFA) to compare the metabolic states of cells during growth and adenovirus production and to optimize culture media according to the metabolic demand of the cells in terms of glucose and glutamine concentrations. This allowed obtaining a higher maximum cell concentration and increased adenovirus production by minimizing the production of metabolites that can have an inhibitory effect on cell growth. We have proposed a stoichiometric equation for adenovirus synthesis. MFA results allowed determination of how these changes in composition affected the way cells distribute their nutrient resources during cell growth and virus production. Virus purification was successfully achieved using chromatography and Aqueous Two-Phase Systems (ATPS).     

Development and optimization of an adenovirus production process

Adenoviral vectors have a number of advantages such as their ability to infect post-mitotic tissues. They are produced at high titers and are currently used in 28% of clinical protocols targeting mainly cancer diseases through different strategies. The major disadvantages of the first generation of recombinant adenoviruses are addressed by developing new recombinant adenovirus vectors with improved capacity and safety and reduced inflammatory response. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. HEK-293 complementing cell line physiology, metabolism and viral infection kinetics were studied at small scale to identify optimal culture conditions. Batch, fed-batch and perfusion culture modes were evaluated. Development of new monitoring tools (in situ GFP probe) and quantification techniques (HPLC determination of total viral particles) contributed to acceleration of process development. On-line monitoring of physiological parameters such as respiration and biovolume of the culture allowed real-time supervision and control of critical phases of the process. Use of column chromatographic steps instead of CsCl gradient purification greatly eased process scale-up. The implementation of the findings at large scale led to the development of an optimized and robust integrated process for adenovirus production using HEK-293 cells cultured in suspension and serum-free medium. The two-step column-chromatography purification was optimized targeting compliance with clinical material specifications. The complete process is routinely operated at a 20-L scale and has been scaled-up to 100 L. Scale-up of adenoviral vector production in suspension and serum-free medium, and purification according to regulatory requirements, are achievable. To overcome metabolic limitations at high cell densities, use of perfusion mode with low-shear cell retention devices is now a common trend in adenovirus manufacturing. Further process improvements will rely on better understanding of the mechanisms of virus replication and maturation in complementing host cells.