Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin

Journal Pre-proof Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin Hubert Schwarz, Ye Zhang, Caijuan Zhan, Magdalena Malm, Raymond Field, Richard Turner, Christopher Sellick, Paul Varley, ´ Johan Rockberg, Veronique Chotteau

PII:

S0168-1656(19)30953-8

DOI:

https://doi.org/10.1016/j.jbiotec.2019.12.017

Reference:

BIOTEC 8573

To appear in:

Journal of Biotechnology

Received Date:

15 January 2019

Revised Date:

11 December 2019

Accepted Date:

26 December 2019

Please cite this article as: Schwarz H, Zhang Y, Zhan C, Malm M, Field R, Turner R, Sellick C, Varley P, Rockberg J, Chotteau V, Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.12.017

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Research Article

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin

Hubert Schwarz 1,2,3

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Ye Zhang 1,2,3 Caijuan Zhan 1,2

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Magdalena Malm 2,4

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Raymond Field 5 Richard Turner 5

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Christopher Sellick 5,6

Johan Rockberg 2,4

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Véronique Chotteau 1,2,3

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Paul Varley 5

School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Dept. of Industrial

Biotechnology, Cell Technology Group (CETEG), Royal Institute of Technology (KTH), Stockholm, Sweden

Wallenberg Centre for Protein Research (WCPR), Stockholm, Sweden

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2

3

Centre for Advanced Bioproduction by Continuous Processing (AdBIOPRO), Stockholm, Sweden

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School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Dept. of Protein

Science, Royal Institute of Technology (KTH), Stockholm, Sweden 5

Biopharmaceutical Development, MedImmune, Cambridge, UK

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Kymab, Cambridge, UK

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Correspondence: Véronique Chotteau, Dept. of Industrial Biotechnology, Cell Technology Group (CETEG), Royal Institute of Technology (KTH), Roslagstullsbacken 21, 114 21 Stockholm, Sweden E-mail: [email protected]

Highlights

A novel small-scale perfusion bioreactor system for animal/human cells with working

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

volumes <250 mL was developed.

HEK293 cells expressing Erythropoietin were utilized in a high cell density perfusion

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

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process up to 77 days in culture.

Densities above 80 x 106 HEK293 cells/mL were achieved with a perfusion rate of 10

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pL/cell/day using nutrient enriched feed medium.

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Abstract

Process intensification in mammalian cell culture-based recombinant protein production has been

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achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be

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disadvantageous compared with human cell lines. In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and

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agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell

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lines, such as HEK293 can be used for intensified perfusion processes.

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Abbreviations: CHO, Chinese Hamster Ovary; HEK293, Human Embryonic Kidney 293; rhEPO,

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recombinant human Erythropoietin; IgG, Immunoglobulin G, TFF, Tangential flow filtration; ATF, Alternating tangential flow filtration; HF, Hollow fiber filter; CSPR, Cell specific perfusion rate; EDR,

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Energy dissipation rate

Keywords: Chinese Hamster Ovary (CHO) cells, Erythropoietin, High cell density culture, Human

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Embryonic Kidney 239 (HEK293) cells, Perfusion process, Small-scale bioreactor

1 Introduction

Interest in perfusion processes in the manufacturing of biopharmaceuticals has increased in recent

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years, largely due to higher volumetric productivities and the potential to reduce capital expenditures compared to legacy fed-batch processes (Konstantinov and Cooney, 2015). In perfusion systems, the cells are commonly retained in the bioreactor whereas cell-free supernatant containing the protein of interest is separated from the cell suspension and continuously removed. Owing to a continuous renewal of the medium in the bioreactor, a high cell density and prolonged process time can be achieved in a perfusion process, potentially resulting in a high volumetric productivity of the

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protein of interest. Thus, the bioreactor size and correspondingly investment spending can be drastically reduced in comparison to fed-batch mode. Also improved product quality attributes have been previously described in perfusion operation (Lipscomb et al., 2005; Zhuang et al., 2017). Alternatively, the biopharma field applies also perfusion in hybrid culture modes where e.g. the product of interest is retained by an ultrafilter or perfusion is applied in the seed bioreactor (Clincke et al., 2013a; Yang et al., 2014).

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The development of a perfusion process can generally be seen as a time and resource-consuming task. The lack of small-size perfusion systems for process characterization and medium screening is an

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impediment to both the development and implementation of continuous production processes for

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biologics in today’s industry. In an effort to reduce the expenditures for process development, it is important to have access to a screening system for small-scale operations. For this purpose, semi-

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continuous operated screening systems, such as perfused shake flasks (Fernandez et al., 2008), spin tubes (De Jesus et al., 2004; Villiger-Oberbek et al., 2015; Gomez et al., 2017) and automated micro-

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scale bioreactor systems (Lyons, 2016; Kreye, 2015) are operated with renewal of the medium for instance once per day and not in a continuous mode. These systems enable the operation of multiple

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parallel cultures in a minimized working volume, and therefore can be utilized as an inexpensive tool to generate large amount of information. However, as the culture medium is discontinuously renewed, these models are not simulating conditions of a real perfusion process. The discontinuous medium renewal causes large variations of the metabolite and nutrient concentrations, which result

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in a less accurate mimic of true continuous mode due to higher fluctuations of the kinetic rates or metabolic states. Discrepant results using shake flask cultures have also been described due to a lack of control of process parameters, for example the pH, owing to the fact that metabolites can accumulate in semi-perfusion (Fernandez et al., 2008). Furthermore, dissolved oxygen (DO) limitations in such scale-down models can lead to lower maximal cell densities than in more sophisticated continuous perfusion bioreactors (Gomez et al., 2017). In light of these disadvantages,

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fully continuous small-scale perfusion systems supporting high cell densities are better suited for process development. In the present work, we developed such a system using commercial parallel bench-top bioreactors. The cell retention device plays a crucial role in a perfused system. Very high cell densities of Chinese Hamster Ovary (CHO) cells, well beyond 108 cells/mL, have been achieved and maintained for longterm operations only by using tangential flow filtration devices (Clincke et al., 2013a,b) and perfusion

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bioreactors with entrapped cells in a matrix (Zhang et al., 2015). Tangential flow filtration (TFF) using hollow fiber filter cartridges operated as such or with alternating tangential flow (ATF) have

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been proven as a method of choice for cell separation in perfusion processes for recombinant protein

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production (Clincke et al., 2013a; Yang et al., 2014; Lin et al., 2017; Karst et al., 2016). The scalability of most cell retention devices is very limited and can become a challenge especially for industrial

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scale processes (Woodside et al., 1998; Voisard et al., 2003), but also for perfusion cultures in minimized working volumes. Due to the fact that hollow fiber membranes can support high cell

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densities, our novel perfusion system was developed by the incorporation of either a TFF or ATF system.

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Antibody producing CHO cells were used for the development of this perfusion system to achieve very high cell densities (108 cells/mL) in a stirred tank bioreactor with small working volume (<250 mL). CHO cells are the workhorses of the biopharmaceutical industry, however human cell lines, such as Human Embryonic Kidney (HEK293) cells have been explored and used in manufacturing

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processes as well. Therapeutic proteins produced by human cell lines possess more human-like posttranslational modifications than proteins expressed from rodent cell lines such as CHO cells, and thus their therapeutic efficiency and safety can be improved (Walsh and Jefferis, 2006; Ghaderi et al., 2010). Human cells can also potentially support expression of difficult-to-produce proteins where CHO cells are suboptimal (Johan Rockberg, personal communication). So far, only five therapeutic proteins produced from HEK293 cells have been registered for commercial distribution by the FDA

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and EMA (Dumont et al., 2016), but with a growing interest in the utilization of human cell lines for biopharmaceutical manufacturing, it is expected that a much wider range of products derived from human expression systems will enter the market in the future (Bandaranayake and Almo, 2014). The aim of this work was to demonstrate that HEK293 cells can be used as an alternative to the predominant CHO cell lines in the biopharmaceutical industry for high cell density perfusion cultivations. We showed that a HEK293 culture stably expressing recombinant human

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Erythropoietin (rhEPO) can be successfully maintained in our small-scale perfusion system for a period of 11 weeks. Furthermore, we investigated the effect of several key process parameters, such

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as the cell density, the temperature and the perfusion rate on the overall performance of the culture.

2 Materials and Methods

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2.1 Cell lines and media

In this study, CHO DP12 clone#1934 cells (ATCC CRL-12445™) and CHO-M cells (Selexis) expressing

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IgG (Immunoglobulin G) and a HEK293F cell line producing rhEPO (KTH in-house developed) were used.

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CHO cells were thawed in BalanCD CHO Growth A medium (Irvine Scientific), supplemented with 4 mM glutamine and cultivated in shake flasks at 37 °C, 5 % CO2 atmosphere and 100 rpm agitation prior to their inoculation in the bioreactors. The same protocol was applied for HEK293 cells, which were grown in BalanCD HEK293 medium (Irvine Scientific), supplemented with 4 mM glutamine and

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50 mg/L puromycin dihydrochloride (Sigma-Aldrich) to provide selective pressure. The medium used in the perfusion experiments was BalanCD CHO Growth A medium without glucose supplemented with 100 mg/L streptomycin and 60 mg/L penicillin G (both Sigma-Aldrich) for the CHO cell cultures. For the HEK293 cell cultures, the feed medium, called Feed A, was BalanCD HEK293 medium supplemented with 5 % BalanCD HEK293 Feed (Irvine Scientific). An enriched variant of this latter mix, called Feed B, consisting of 1.3x concentrated BalanCD HEK293 medium

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supplemented with 10 % BalanCD HEK293 Feed, was also used as indicated in the text. A glucose and glutamine targeted feeding strategy was applied to all the perfusion runs. With this strategy the concentrations of these nutrients in the feed were adjusted to correspond to the expected cell need and to keep the formation of their byproducts lactate and ammonium below 20 mM and 4 mM, respectively.

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2.2 Perfusion bioreactor design The study was conducted in 350 mL (total volume) glass stirred tank DASbox bioreactors

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(Eppendorf) with a working volume of 230 mL. The hollow fiber filter (HF) was a Xampler™ CFP-2-

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E-3MA microfiltration cartridge (GE Healthcare) with 0.2 μm pore size and 110 cm2 membrane area. The perfusion runs were performed either in tangential flow filtration (TFF) or alternating tangential

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flow (ATF) mode. In TFF mode, the cell suspension was pumped by a peristaltic pump (Alitea) through a PharMed BPT tube (Saint-Gobain) into the HF with a flow rate of 50 mL/min. For the ATF

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mode, the HF was connected to an XCell™ ATF 2 system (Repligen) and the flow rate recirculation was set to 200 mL/min. A dip tube with 4 mm internal diameter was used in both cases to pump the

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cell broth into the HF.

2.3 Characterization of the gas-liquid transfer in the bioreactor The oxygen transfer in the bioreactor was evaluated for different impeller setups. The bioreactors

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were equipped with 3-Blade marine impellers, 6-Blade Rushton-type impellers or a combination of two impellers. For each impeller configurations, the volumetric oxygen transfer coefficient (kLa) in the bioreactor was determined by the dynamic gassing-out method (Van Suijdam et al., 1978), where the headspace of the reactor was aerated with a constant flow of air (0.35 vvm) after an initial “stripping-out” procedure with N2. Additionally, the effect of sparging the liquid with air at 0.035 vvm was tested. The impeller speed was set to 400 rpm for all configurations.

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The maximum energy dissipation rate (EDR) for the different impeller types in stirred tank bioreactors was calculated as following: 𝜀𝑚𝑎𝑥 = 𝐸 × 𝑁 3 × 𝐷 2 × 𝜌

[Eq. 1]

where E is a non-dimensional constant depending on the impeller type, N is the stirring speed, D is the impeller diameter and 𝜌 is the density.

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2.4 Perfusion processes Bioreactors were inoculated at a cell density of 1 x 106 cells/mL. After an initial 1-day batch mode,

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perfusion operation was started by a continuous supply of a feed medium into the reactor and

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removal of harvest from the permeate side of the HF at 0.5 reactor volume/day (RV/day) using the peristaltic pumps from the DASGIP MP8 module (Eppendorf). The working volume in the bioreactor

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was kept constant at 230 mL by equal inflow and outflow rates with calibrated peristaltic pumps and careful monitoring of the ingoing and outgoing weights for adjustment of the flow rates. In this work,

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a perfusion rate that correlates with the cell density was chosen. This was achieved by applying a constant cell specific perfusion rate (CSPR), which is the amount of medium that is perfused per cell

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per day. Based on previous work with these CHO cells, a CSPR of 50-60 pL/cell/day was selected (Zhang et al., 2015). This was also applied to HEK293 perfusion cultures as a starting value. Cell bleeding was applied by a continuous removal of cell suspension from the bioreactor in order to stabilize the cell density at a given level. The selected bleed rate was equivalent to the growth rate in

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order to maintain a constant cell density. The temperature was maintained in all experiments at 37 °C, except in run HEK#2, where it was reduced to 33 °C on day 28. The pH was controlled at set point 7 by automatic additions of 0.5 M Na2CO3 or CO2 into the headspace of the reactor. The DO set point was 40 % and maintained by a continuous flow of air enriched in O2 or N2 into the headspace of the bioreactor. Additionally, O2 was added with 0.0035 - 0.07 vvm through a stainless steel L-shaped

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orifice sparger (Eppendorf) into the liquid as the oxygen consumption rate in the system increased and headspace oxygenation became limited.

2.5 Process analytics Daily samples taken from the bioreactor were analyzed with a Bioprofile FLEX analyzer (Nova Biomedical) to determine the viable and total cell density, viability, pH, pCO2, osmolality and the

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concentrations of glucose, lactate, glutamine and ammonium. Amino acid analysis was performed based on a Waters UPLC Amino Acid Analysis applications kit

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using the vendor’s protocol; briefly: Cell culture supernatants were filtered through 10 kDa filters

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and filtrates were diluted using HPLC grade water to appropriate concentrations. Amino Acid Standard (Waters) was diluted for use as a standard curve. Diluted samples and standards were

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combined with internal standard Norvaline (Sigma-Aldrich) and AccQ Tag Derivatization Kit reagents (Waters). Samples were run on an Acquity UPLC system (Waters) using an AccQ-Tag Ultra

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RP Column 130 Å 1.7 μm, 2.1 mm, 100 mm (Waters). Data were analysed using EMPOWER software (Waters).

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IgG concentrations were analyzed by protein A affinity HPLC. A POROS A column (Thermofisher Scientific) with a Supelco ColumnSaver 0.2 μm (Sigma-Aldrich) pre-column filter was equipped in an Alliance HPLC system (Waters). IgG signals were obtained at 214 nm with a photodiode array detector PAD 2996 (Waters). Concentrations of rhEPO were quantified with an EPO human ELISA kit

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(Thermofisher Scientific). According to a reference standard, a factor of 7.96 was used to convert IU/mL into µg/L.

The cell specific consumption and production rates of nutrients, metabolites and product were calculated as previously described (Clincke et al., 2013a,b). The sieving coefficient was calculated as the ratio of product concentration in the harvest PH to product concentration in the bioreactor PB: 𝑃

𝜎 = 𝑃𝐻 × 100 (%) 𝐵

[Eq. 2]

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3 Results and Discussion 3.1 Development of a small-scale perfusion system for high cell density cultures In the first part of this study, a novel perfusion system for small-scale operations in fully continuous mode was established, aiming at providing a reliable method for the achievement of very high cell

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densities of up to 108 cells/mL in a very low working volume stirred tank bioreactor.

3.1.1 Bioreactor evaluation

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Commercially available parallel screening systems such as DASGIP and DASbox (Eppendorf) are

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commonly applied as scale-down models for process development of batch and fed-batch processes, however in this study we re-purposed them by using an external HF as cell retention device to

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develop a perfusion platform that is able to support high density cultures of mammalian cells. Perfusion runs using CHO cells were performed in the bioreactors using the TFF and ATF setup. The

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cell density was increased to a target value of 80-100 x 106 cells/mL, while the perfusion rate was proportionally increased, once a day based on the cell density measurement, to maintain a constant

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average CSPR between two sample points. In the TFF run, a cell density of 101 x 106 cells/mL was reached with a viability above 90 % (Figure 1A). A similar profile was shown for the ATF run, where a cell density of 80 x 106 cells/mL was achieved with a viability of 89 % (Figure 1B). Achieving a high cell density above 80 x 106 cells/mL however altered the process performance in

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these runs. The partial pressure of CO2 reached critical values with up to 15 kPa in the bioreactor due to an insufficient removal of CO2 out of the liquid phase. The DO dropped below the set point at cell density >80 x 106 cells/mL, indicating that the oxygen transfer in the system became limited even for high sparged oxygen flow rates. Excessive foaming in a small headspace volume occurred and was highly detrimental for the process since foam could easily block the off-gas filter for outgoing air. As a consequence, the maintenance of a high cell density became challenging and the process runs had

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to be interrupted shortly after reaching the target cell density. To overcome these problems, further process optimization was carried on as described below in Section 3.1.3.

3.1.2 Cell separation system To establish a well-functioning perfusion platform process, one relies on a robust cell retention device. Therefore, the effect of the filtration mode (TFF/ATF) on the important process parameters

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cell viability and product sieving was evaluated. In preliminary experiments it was observed that the recirculation flow rate in the TFF bioreactor had

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a detrimental impact on the viability of the culture (data not shown). A viability above 90 % was only

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maintained with a flow rate ≤50 mL/min, a value that was later adopted in the TFF perfusion run. As reported by Wang et al. (2017), a peristaltic pump can account for the major loss in cell viability due

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to the high shear forces that arise in the pump tube. Conversely, the diaphragm pump of the ATF system provides a low shear device and potentially improved process performance.

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For both types of filtration systems, a sufficiently high viability was maintained in the perfusion runs after fine-tuning of the recirculation flow rate. However, a different degree of product recovery was

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observed between TFF and ATF, as monitored by the product sieving coefficient. In the ATF system, the majority of the product passed the filter membrane and was harvested with a sieving coefficient of 91-99 % throughout the culture (Figure 1B), while in TFF mode the sieving steeply decreased to 19 % (Figure 1A). The recirculation flow rate of 50 mL/min in the TFF was 4-fold lower than in the

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ATF system and thus potentially too low to create a shear sufficient to avoid the rapid creation of a gel layer clogging the filter membrane. This trapped significant amounts of the product of interest inside the bioreactor. From this observation, it was concluded that the ATF system was more advantageous than the TFF in this small volume bioreactor since higher recirculation flow rates could be applied to minimize the effect of membrane fouling. Using a pump creating a lower shear stress in the pump head could circumvent the limitation of the low flow rate in the TFF and thus potentially

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give comparable results to the ATF. In ATF mode, rapid oscillations of the liquid level occurred in the bioreactor due to the alternating in -and outflow of the cell broth. Although the bioreactor size was slightly undersized for the present ATF system, no detrimental effects on the cell growth, viability or other process parameters were observed. All the subsequent perfusion runs were performed with the ATF system.

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3.1.3 Optimization of the oxygen transfer in the bioreactor CHO cell perfusion runs achieved a cell density beyond 80 x 106 cells/mL, however at this very high

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cell concentration the process became limited in the oxygen transfer and carbon dioxide removal. A

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high stirring speed (400 rpm) and high oxygen flow rate (up to 0.07 vvm sparging) was applied to compensate for the increased oxygen demand. This led to excessive foam generation, risking

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overflowing the bioreactor. Sparging of oxygen and intensive stirring provoke stress in the cells due to high hydrodynamic shear forces that arise from bubble rupture and turbulent agitation (Hu et al.,

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2011). The applied aeration and agitation strategy was rather well tolerated by the robust CHO cells. However, when this process setup was applied to a HEK293 cell perfusion culture, it was not possible

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to achieve comparably high cell densities with satisfying viability (data not shown). A study was carried out to improve the bioreactor mixing and oxygen transfer to support the high oxygenation demand of a high-density culture without damaging the HEK293 cells.

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Table 1. Comparison of impeller configurations in the bioreactor where 'bottom' and 'top' refer to impeller(s) placed at the bottom and the top of the bioreactor drive shaft. The marine impellers were positioned such that they were pumping the liquid downwards.

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Design

Setup 2 Marine impellers (top and bottom)

Setup 3 Rushton impeller (bottom)

Setup 4 Rushton impeller (top) Marine impeller (bottom)

kLa [h-1]

2.5

7.3

4.2

19.5

kLa [h-1]

6.9

14.7

7.0

29.2

Average  [d-1]

NM a

0.58  0.07

0.59  0.05

Viability [%] b

NM

97.6  1.0

98.5  0.1

98.5  0.1

EDR [W/m3] c

240

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3,973

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0.60  0.01

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with sparging

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without sparging

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Setup 1 Marine impeller (bottom)

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NM, not measured Viability on last day c EDR estimation based on Eq. 1 for 400 rpm stirring speed, 30 mm impeller diameter and an E value of 14.9 for the Rushton-type impeller and 0.9 for the marine impeller (similarity assumed to high-efficiency impeller HE-3) (Zhou and Kresta, 1996) a

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b

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Different impeller configurations of Rushton-type and marine impellers, presented in Table 1, were compared and characterized by their oxygen transfer rate coefficient (kLa). Setup 1 was the

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configuration that was used in the previous CHO cell culture runs and generated the lowest kLa value. The highest kLa was achieved with a Rushton-type impeller positioned at the top and a marine impeller mounted at the bottom of the drive shaft (setup 4). Thus, the mass transfer in this dual impeller configuration was improved by a factor of 7.8 compared to the original setup. Sparging the

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bioreactor in addition with air at one tenth of the flow rate of the overhead aeration resulted in increased kLa values in all configurations. The degree of influence from the impeller setup on the oxygen transfer slightly changes when sparging is applied. The selection of a dual impeller setup, however, is still in favor over a single impeller system due to an improved gas exchange through the headspace. As expected, the results also show that the Rushton-type impeller resulted in a higher energy dissipation, i.e. more turbulent flow compared to the marine impeller, which generated a 1.7-

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fold kLa increase in setup 3 compared to setup 1 and an EDR of 3,973 W/m3 instead of 240 W/m3. Interestingly, the kLa was similar in both setups when sparging was applied, possibly due to the higher residence time of bubbles in the liquid, which was caused by the different fluid flow from the marine impeller. The effect of the different impeller configurations on the cell growth and viability was studied in 4day batch cultures with HEK293 cells. No difference in the growth rate and viability of the cultures

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were observed between the different setups (see Table 1), hence stress due to turbulence was not an issue for the cells in the tested configurations. This indicates that the EDR created by the Rushton-

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type impeller did not cause lethal damage to this cell line (the EDR for setup 4 could not be

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theoretically calculated by Eq. 1 due to the complex impeller setting). The combination of a marine impeller at the bottom and a Rushton-type impeller at the top (setup 4), achieving the highest kLa,

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was therefore selected and used in the subsequent perfusion runs. In setup 3, the EDR was in the same range as the EDR in a 22,000 L bioreactor stirred at 140 rpm with a Rushton impeller, while the

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EDR of the rupture of a 6.3 mm diameter bubble is two orders of magnitude larger (Godoy-Silva et al., 2009).

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It has been reported that sensitivity with respect to hydrodynamic shear forces differs widely among different animal and human cell lines (Mollet et al., 2004). Although no study directly compares the shear sensitivity of CHO and HEK293 cells, the effect of shear stress on various human cell lines, with potentially similar properties to HEK293, was characterized in previous studies. Mollet et al. (2008)

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demonstrated that a human leukemia cell line is more sensitive to the EDR compared to CHO cells. While the CHO cells in our initial non-optimized perfusion system tolerated the operating conditions, the HEK293 cell line highly suffered from lethal effects. Besides the optimization of the mass transfer in the bioreactor, cell engineering or laboratory evolution could be carried out to create HEK293 cells with greater shear resilience.

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Rushton-type impellers are not as commonly used as marine impellers for the cultivation of mammalian cells and have been rarely reported as being used for HEK293 cells (Junker, 2004). This applies in particular for large-scale cell cultures, however in the present small-scale bioreactors, this impeller exhibited a good performance for the cultivation of HEK293 cells, probably due to the small impeller diameter.

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3.2 HEK293 perfusion cultures Perfusion cultures of HEK293 cells producing rhEPO were then performed in this novel small-scale

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perfusion bioreactor system including the optimized impeller setting of Section 3.1.3. The first step

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was to demonstrate the capability of this system to maintain a very high cell density of HEK293 cells. Then the influences of cell density, process temperature and perfusion rate on the performance of

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3.2.1 High cell density steady-state

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the culture were studied.

In run HEK#1 (Figure 2A), the cell density was at first maintained by cell bleeds at around 20 x 106

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cells/mL for a period of 14 days with a viability above 90 %. Afterwards, the cell concentration was progressively increased in small steps of 2 to 5 days, from 20 to 30, then 50 and 70 x 106 cells/mL with careful monitoring of the cell viability and growth in the system. This was realized by interrupting the bleed function for 1-2 days, i.e. reduction of the bleed rate to 0 RV/day, as shown in

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Figure 2C. Finally, the consistency of this process was confirmed at a density of 70-80 x 106 cells/mL with a sustained high cell viability ≥95 % for one week. Thanks to the optimization of the oxygen transfer in the bioreactor described in the previous section, cell densities comparable to the CHO cell trial runs were achieved. CO2 was sufficiently removed from the liquid phase with peak pCO2 values of 14 kPa in the culture (data not shown). Run HEK#2 was then conducted to demonstrate the reproducibility of this perfusion process for a high cell density.

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In run HEK#2 (Figure 2B), the cell density was increased to 80 x 106 cells/mL within 11 days (i.e. without a lower cell density stage) and maintained for a period of 66 days with a viability of 95 % at the end of the run. On day 29, mild hypothermia at 33°C was applied to the culture to investigate if this could improve the process performance. The cell density and viability remained stable after lowering the temperature, while cell growth was almost completely arrested at 33 °C. The respective data for the growth rates and applied bleed rates are shown in Figure 2D. A 5-fold reduction of the

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perfusion rate from 4.2 to 0.8 RV/day, supported by nutrient enriched medium (Feed B) as compensation for the reduced perfusion rate, was stepwise applied from day 35 to 45. As shown in

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for a very low CSPR of approximately 8-12 pL/cell/day.

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Figure 2B, the viability was maintained ≥90 %. This demonstrated the robustness of the process even

The present results show for the first time a high density HEK293 cell culture at >80 x 106 cells/mL

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maintained in a long-term perfusion process with a peak cell density of 111 x 106 cells/mL. Cell densities up to 93 x 106 cells/mL have been previously reported for HEK293 cells in a perfusion

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culture, however not maintained for long-term at this level due to difficulties in maintaining the pH

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and DO at the set point (Kyung et al., 1994).

3.2.2 Production of rhEPO

The effect of the viable cell density on the volumetric productivity of rhEPO was studied in run HEK#1 (Figure 2A). At densities of 15-25 x 106 cells/mL, the average volumetric productivity was 128.9 (±

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26.3) mg/L/day while for high densities of 70-80 x 106 cells/mL it increased to 392.4 (± 47.8) mg/L/day (Figure 3A). The results illustrate that the volumetric productivity linearly increased with the cell density, demonstrating that a high cell density is preferred to maximize recombinant protein production. The high volumetric productivity was later confirmed in run HEK#2 for a cell density of 70-90 x 106 cell/mL with 460.9 (± 76.4) mg/L/day.

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Applying mild hypothermia to 33 °C in run HEK#2 showed in average slightly lower productivities of 370.7 (± 79.6) mg/L/day. It is worth noticing that loss of product was reduced due to lower bleed rates for the 33 °C culture and hence an acceptable compromise for the lower productivity. Lin et al. (2015) observed a 1.5-fold increase of the protein yield in transiently transfected HEK293S cells after decreasing the temperature to 33 °C. Productivity enhancement by hypothermia is known to be cell specific (Mason et al., 2014), and for the cell line used in our study, reducing the temperature did not

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result in productivity enhancement. These results were obtained in perfusion culture HEK#2 at constant CSPR (50-60 pL/cell/day). An

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objective in the development of industrial perfusion processes is to minimize the perfusion rate in

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order to keep operating costs low. Key criteria to adopt a low perfusion rate are to maintain the cell specific productivity and viability high. Application of a low CSPR, however, requires well-balanced

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media to support an intensified perfusion process. As described in the previous section, a concentrated medium with a higher proportion of feed supplement was used to perfuse the

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bioreactor from initially 4.2 RV/day down to 0.8 RV/day. At this extremely low perfusion rate, the cell specific productivity dropped to 3.1 (± 0.6) pg/cell/day (Figure 3B). This represented a decrease

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of 35 % and 50 % in average compared to the high CSPR culture at 33°C and 37 °C, respectively. This decrease concurred with the altered metabolic profiles, showing a significant reduction in the uptake of amino acids presented in Section 3.2.3. A stable cell density with high viability above 90 % was maintained during that period, while an 81 % reduction of the total medium consumption was

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achieved in this process. This can be considered as an important cost saving, not only for the production and logistics around media storage, but also in terms of smaller harvest volumes generated for the subsequent downstream operations. To fully exploit the benefit of a very low CSPR culture, further media optimization is required to maintain the cell specific productivities at a consistently high level. We suggest that the use of concentrated powder medium is a feasible approach to decrease the CSPR in the culture, however this method is limited by the poor solubility

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of certain components and the vast increase of the osmolality, particularly due to the salts, which should be reduced. Therefore, a modification of salt composition would be necessary to maximize the benefits of such a method. For the commercial production of rhEPO, CHO cells are usually selected as host cell line (Lee et al., 2012). Previous reports have therefore primarily focused on studying the effect on rhEPO production in CHO cells under various conditions, including reduced temperature (Yoon et al., 2003; Yoon et al.,

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2005; Ahn et al., 2008), lactate supplementation (Choi et al., 2007), variations in DO (Rastelli et al., 2006), etc. Contrary to the present experimental results, the cell specific productivities generally

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increased at reduced temperatures in batch cultures of CHO cells with 7.4 pg/cell/day at 32.5 °C

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(Yoon et al., 2005), 8.4 pg/cell/day at 33 °C and 11.8 pg/cell/day at 30 °C (Yoon et al., 2003). Similar observations were made in perfusion mode, where the productivity increased from 1.9 pg/cell/day

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at 37 °C to 9.2 pg/cell/day at 32 °C (Ahn et al., 2008). The cell specific rhEPO productivity for the HEK293 cell line in the present study was 6.7 pg/cell/day for low cell density and 6.2 pg/cell/day for

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high cell density (Figure 4C), which were in a comparable range as the productivities described in the CHO cell studies mentioned above. Hence HEK293 cells can be seen as an alternative expression

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host for rhEPO production. Furthermore, process intensification through high cell density perfusion has led here to overall enhanced volumetric productivities.

3.2.3 Cell metabolism

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The cell specific consumption and production rates of proteinogenic amino acids are given in Figure 4A, where the data of four distinct experimental conditions are represented: cell density <25 x 106 cells/mL -37 ˚C -high CSPR; >70 x 106 cells/mL -37 ˚C -high CSPR; >70 x 106 cells/mL -33 ˚C -high CSPR; >70 x 106 cells/mL -33 ˚C -low CSPR. The consumption of the amino acids remained stable after the steady-state shift from medium (15-25 x 106 cells/mL) to high (70-90 x 106 cells/mL) cell density, except for proline, alanine (both reduced consumption) and glycine (increased production). These

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data indicate that no substantial shift in the cell metabolism occurred from this 4-fold increase of the cell density. This can be attributed to the constant cell specific perfusion rate (50-60 pL/cell/day) that was applied in these perfusion runs. The results are in accordance with recent work showing a stable metabolic profile across a wide cell density range in perfusion (Zamani et al., 2018). During the perfusion culture, glutamine was not consumed after the transition from batch to perfusion mode (at day 2), although it was present in the feed medium.

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Shifting the temperature from 37 °C to 33 °C, caused a metabolic shift towards slightly decreased amino acid consumption, except for cysteine. Despite the reduced cell growth at lower culture

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temperature, amino acid consumption remained rather high, possibly due to an increase in cell size

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and thus an increased demand for nutrients. Furthermore, the amino acids alanine, tyrosine, glutamate and glutamine, which were initially consumed in small amounts, were produced under

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mild hypothermia. Alanine, glutamate and glutamine release are possible pathways for ammonium detoxification in cells, which probably occurred in the present case (Dadsetan et al., 2013; Watford,

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2000). Since ammonium production did not increase, larger amounts of ammonium were excreted from cells in the form of alanine, glutamine and glutamate. Additionally, a lower glucose consumption

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was observed under mild hypothermia, which possibly leads to an increased demand of amino acids for compensation.

The CSPR decrease to an extremely low level of 8-12 pL of Feed B/cell/day significantly altered the metabolism of the cells in the culture by a 45-70 % reduction of the cell specific consumption rates.

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Feed B, which was Feed A enriched by concentrated medium and increased feed supplementation, resulted in 1.3 to 1.5-fold increased amino acid delivery per volume compared to the original Feed A. Thus, the relative proportions of the amino acids among each other were more or less maintained between Feed A and Feed B. Figure 4B represents the portion (in percent) of the amino acid, which has been consumed in comparison of the amount of these metabolites supplied from the medium fed in the perfusion. This portion increased above 90 % for eight amino acids, valine, leucine, serine,

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asparagine, arginine, isoleucine, cysteine and alanine, which led to very low residual concentrations of these in the culture (see Figure S1). No complete depletion of the amino acids occurred, however. Productions of alanine and tyrosine were reverted to consumption for the low CSPR condition, and proline consumption completely stopped to be slightly produced. The concentrations of other amino acids (aspartate, lysine, threonine, phenylalanine, tryptophan and histidine) did not noticeably change in the culture, nevertheless their cell specific consumption also decreased significantly, e.g.

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by 70 % for tryptophan (see Figure 4A). This metabolic shift can clearly be attributed to the low CSPR. A more than 5-fold reduction of the

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CSPR resulted in a decreased availability of these nutrients per cell. It is probable that the amino acid

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consumption rate can be described by a Michaelis-Menten model. This implies that at a very low availability of an amino acid, this rate is linearly increasing with its concentration. Thus, the lower is

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the amino acid concentration, the lower is its consumption rate, while above a given concentration, the saturation is reached and the consumption rate does not depend on the amino acid concentration.

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In spite of the low perfusion rate for a very high cell density and consequently reduced metabolism, the culture remained highly stable with respect to maintenance of the cell density and viability,

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however the rhEPO productivity decreased (Figure 4C). These results suggest that despite no apparent depletion of the essential amino acids, the cells might have favored a metabolic pathway, which reduced the rhEPO production.

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4 Conclusions

A small-scale perfusion bioreactor system using hollow fiber cartridges for tangential flow filtration was developed. Very high cell densities above 100 x 106 cells/mL of mammalian/human cells were achieved in this system in a minimized working volume below 0.25 L. To our knowledge, these processes were the first achieving very high cell densities in such a small volume. Operating with a low working volume enables a significant reduction of the medium consumption, which is a major

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cost driver in a perfusion process (Lim et al., 2018). The use of a stirred tank-controlled bioreactor with an ATF system for the cell retention in small volume has proven to be a suitable device for scaledown models. The only factor, which was not well scaled-down was the over-dimensioned bulb volume of the ATF2 system. An issue that is common among filtration-based retention devices, and was also observed in this study, was fouling of the filter membrane. This was solved by a manual exchange of the hollow fiber cartridge when product sieving was severely impaired, in our study

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typically after around 30 days of perfusion. Fouling is described as a function of several parameters, such as the recirculation rate, filter flux, cell density and viability (Walther et al., 2019). The low

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recirculation rate and high cell density mainly contributed to the filter fouling rate. A higher

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recirculation rate in the ATF could be beneficial to reduce this fouling, however it was not tested. This perfusion bioreactor system is highly valuable for process development, e.g. for screening in

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perfusion mode to study process parameters and culture media formulations. The utilization of multiple parallel screening reactors for perfusion cultures supported by an efficient control system

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has the benefit of substantially reducing medium consumption, simplifying process operations and decreasing the footprint. Altogether, this enables the generation of more experimental data at lower

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costs in a shorter time frame.

In recent years, high cell density perfusion of CHO cells has been profiled as a new avenue for efficient biopharmaceutical production. Our results pave the way towards using other types of cells that are less robust than CHO cells, such as HEK293 cells, today commonly used for transient expression or

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viral vector production, but less frequently used for biopharmaceutical manufacturing by stable expression. We show that HEK293 cells can be stably cultured at >80 x 106 cells/mL for a duration of several weeks and that the volumetric productivity linearly increases with the cell density. These results also show that beyond CHO and PER.C6 cells cultured at very high density, other cell lines are suitable for this type of intensified culture, in particular in view of the fact that the volumetric productivity increases with the cell density. Furthermore, this work provides evidence that a very

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low CSPR of 10 pL/cell/day is sufficient to establish a steady-state culture of HEK293 cells with a high sustained viable cell density and viability. Our study focused on developing a high cell density process in small stirred tank bioreactor, so the medium formulation was not optimized but instead medium enrichment was applied. From this approach, we observed interesting metabolic shifts and potentially efficient usage of some amino acids (over 90% consumption) that could be an objective for all the amino acids. We also observed a

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reduced cell specific productivity that medium improvement could potentially restore. To further explore this finding, we will undertake rational medium optimization, i.e. not only overall enrichment

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of all the components, e.g. by concentrating the medium as presented here, but a specific increase of

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the concentration of selected components, based e.g. on a mathematical modelling approach.

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Author contributions Conceptualization, H.S. and V.C.; Methodology, H.S, R.F., J.R. and V.C.; Investigation, H.S., Y.Z. C.Z., M.M. and C.S.; Result Analysis: H.S.; Writing, H.S., Y.Z., M.M., R.F., J.R. and V.C.; Supervision, J.R., R.F., R.T., P.V., and V.C.

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H.S. Hubert Schwarz Y.Z. Ye Zhang

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C.Z. Caijuan Zhan M.M. Magdalena Malm

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R.F. Raymond Field

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R.T. Richard Turner

P.V. Paul Varley J.R. Johan Rockberg

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V.C. Véronique Chotteau

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C.S. Christopher Sellick

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

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This study was financed by the Knut and Alice Wallenberg Foundation and MedImmune. The authors are thankful to Irvine Scientific for the sponsoring of BalanCD CHO Growth A medium. The authors also thank Selexis SA for the CHO-M cell line.

Conflict of interest

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The authors declare no financial or commercial conflict of interest.

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Figure legends

Figure 1. Profiles of viable cell density (●), viability (◇) and product sieving (△) in CHO cell perfusion test runs. (A) Perfusion run in TFF mode using a CHO-DP12 research cell line achieving 100 x 106 cells/mL. (B) Perfusion run in ATF mode with CHO-M cells. In this run, the process time was

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shortened by inoculating the bioreactor with a higher seed cell density of 5 x 106 cells/mL.

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Figure 2. Cell culture data of ATF perfusion runs performed with recombinant HEK293 cells showing the viable cell density (●), viability (◇) and perfusion rate (△). (A) Run HEK#1 with two steady-states at 15-25 x 106 cells/mL and 70-80 x 106 cells/mL. (B) Run HEK#2 with maintenance of 70-90 x 106 cells/mL at 37 °C until day 28 and subsequent mild hypothermia at 33 °C. From day 36 the perfusion rate was stepwise decreased to 0.8 RV/day, corresponding to a CSPR <12 pL/cell/day. The use of

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nutrient enhanced feed B from day 39 onwards is indicated in the figure by the filled triangles (▲). On day 66, the cell density was increased beyond 100 x 106 cells/mL. Growth rate and specific bleed

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rate from run HEK#1 (C) and HEK#2 (D).

Figure 3. (A) Correlation of the volumetric productivity with the viable cell density. Only data points for a CSPR >40 pL/cell/day are shown. (B) Correlation of the cell specific productivity with the CSPR. Data of perfusion runs HEK#1 (×), HEK#2 – 37 °C (●), HEK#2 – 33 °C (〇) and HEK#2 – 33 °C with nutrient enriched feed B (＋) are shown.

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Figure 4. (A) Cell specific rates of amino acid consumption (positive values) and production (negative values) in HEK293 perfusion processes. The effect of the cell density, temperature and CSPR as described in the legend is shown. (B) The relative consumption of amino acids calculated as

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the ratio of consumed amino acids over supplied amino acids. Error bars are showing the standard deviations of n≥3. (C) Cell specific productivity of rhEPO for the different conditions.

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