Sartoclear Dynamics ® Technology: How to clarify mAb from CHO Fed-Batch in BIOSTAT STR ® 1000 L

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Application ReportCell Clarification

1 Introduction

1.1 Cell Cultivation in a single-use bioreactor BIOSTAT STR ® Sartorius Stedim Biotech offers a wide range of single-use bioreactors from 10 mL to 2,000 L working volume. Their designs are entirely based on proven stirred-tank bioreactor principles ensuring straightfor-ward scale-up from process development to production bioreactors.Within this product range, the BIOSTAT STR ® is a single-use, stirred tank bioreactor for working volumes from 12.5 L to 2,000 L. Due to its conventional cylindrical design and excellent performance characteristics, this bioreactor family is ideal for a wide range of mammalian cell culture processes.

The system is comprised of a stainless steel bag holder, a single-use Flexsafe STR ® bag and a superior control system. The bag holder supports the cultivation chamber and enables heating and cooling via a liquid-filled double wall system. In the DCU controller all essential control tools are implemented. On the touch screen, an user friendly interface shows all the basic functions of the BIOSTAT STR ® such as agitation, gassing, pH and pO2 control. The control tower can easily be connected to the SCADA system MFCS | win for data acquisition and process control.

Sartorius Stedim Biotech has developed a new polyethylene film for single-use products including full traceability of the bag production process – from the resin to the final sterilized bag. This new Flexsafe ® film has a reduced additive package and was especially developed to ensure excel-lent and reproducible cell growth perfor-mance. The Flexsafe STR ® bag is a fully disposable single-use cultivation chamber and contains a pre-installed stirrer with two impellers and a sparger for oxygen transfer and homogeneous culture broth.

1.2 Dynamic body feed filtration for single-use clarification technology Continuous improvements in cell culture media and cell line engineering as well as novel process techniques have resulted in increased biomass concentrations obtained from biopharmaceutical production processes. This leads to more challenging downstream purification processes.

Bertille Lagrange, Sebastian Ruhl, Marcus Peiker, Franziska Faulstich

Currently the most widely applied single-use harvesting technology is depth filtration. More concentrated cell culture supernatants are difficult to process with depth filters, since these filters tend to block at higher biomass concentrations ending up in lower loading capacities. Other cell removal solutions such as single-use centrifugation are avail-able, but as yet lack capacity, which drives costs up and increases process time. Especially for larger single-use bioreactors the use of stainless steel cenrifuges has become essential. Sartoclear Dynamics ® from Sartorius Stedim Biotech is a new single-use technology for harvesting mammalian cell cultures with high cell densities in one single unit operation. This technology draws inspiration from processes used in the blood plasma fractionation industry and is based on the principles of dynamic body feed filtration (DBF). Body feed filtration has proven to be a successful method of solving similar issues in other industries. This robust tech-nology is now available as a harvesting solution for biotechnology applications.Sartoclear Dynamics ® as a ready-to-use clarification system enables single-use processing at volumes of up to 2,000 L. It offers consistent results, ease of use, speed and flexible scalability. In con-sequence, it provides a commercially viable solution that replaces both centrifuges and depth filters in one single step.

Dynamic body feed filtration uses filter aids such as diatomaceous earth (DE) to increase filter capacity, which has a long history of use. DE, diatomite or Kieselgur, is a naturally occurring, soft, siliceous sedimentary rock with a typical particle size of 10 – 200 μm, composed of the shells or exoskeletons of fossilized diatoms (microscopic, single-cell algae). Benefiting from a very porous structure and inert character, DE is often used in the bio-technology industry as an integral and built-in feature in depth filters used during the harvesting stage. In contrast, for Body Feed Filtration the DE is added to the cell culture broth where it plays a dual role: it sieves out sub-micron particles and creates a permeable filter cake that improves filter throughput. The minute filter aid particles provide countless micro-scopic channels that entrap suspended

impurities, but allow clear liquid to pass through without clogging. Diatomaceous earth as a filter aid is available in a wide range of grades, which differ in purity and permeability.Sartorius Stedim Biotech selected Celpure ® C300 as the optimum filtration aid for mammalian cell culture procedures. Celpure ® C300 is a highly purified form of DE that offers an improved operational consistency. It is designed specifically for (bio-)pharmaceutical applications and produced using a patented manufacturing process.

2 Material & Methods

2.1 Bioreactor BIOSTAT STR ® 1000 LFor the process described in this application note, a BIOSTAT STR ® 1000 with Flexsafe STR ® 1000 was utilized for the cultivation of CHO DG44 cells. The bag was equipped with two 3-blade segment impellers and a ring sparger. To reduce exhaust filter blocking a single-use exhaust cooler was used.

2.2 Process and ParametersA mammalian cell culture was used as a model liquid to achieve realistic results during the DBF. The shown model process was a high density fed-batch process with a CHO DG44 cell line. The duration of the industry proven process was 17 days. The viable cell density (VCD) ranged between 25 to 30 × 106 cells/mL at the peak and was higher than 10 × 106 cells/mL at the end. Cell viability at the end of the process was expected to be above 50 %. The expected yield of the produced antibody IgG1 was 7 – 9 g/L. The complete upstream process was performed in single-use equipment from cryo vial to harvest. For determination of cell relevant parame-ters a Cedex HiRes was used. Product concentration was determined by FPLC using membrane adsorbers.

The used cell culture process and cell line are developed by Cellca (now part of Sartorius Stedim Biotech). The medium system comprises four chemically defined media solutions for seed culture, main culture and two feeds. The fully automated feeding process comprised a daily bolus addition of feed medium A and B from day 3 to 16.Additionally a high concentration glucose feed was used to keep the glucose con centration above 3 g/L. The process required several seed culture steps of three days each.

The seed train from 1 mL to 1000 L harvest volume is shown in Figure 1:

Important process parameters for the CHO process in Flexsafe STR ® are shown in the following table:

BIOSTAT STR ® with Flexsafe STR ® 1000 17 d 520 – 1000 L

BIOSTAT STR ® with Flexsafe STR ® 200 3 d 100 L

BIOSTAT ® RM with Flexsafe ® RM20 3 d 10 L

1 L shake flask 3 d 2 × 300 mL

0.5 L shake flask 3 d 150 mL

Cryo Culture 1 mL

Figure 1: Seed train for Flexsafe STR ® 1000 L Cultivation

Table 1: Critical process parameters

Critical process parameter Set point Controlled byTemperature 36.8 °C Double wall system

with cooling | heating liquid

pO2 60 % saturation N2 | Air | O2 gassing rate, agitation rate

pH 7.15 CO2 gassing rate

2.4 Set-up of 1000 L CHO HarvestThe complete process was regulated and controlled via the FlexAct ® platform (see Table 2). CHO cell culture from the Flexsafe STR ® 1000 was transferred into a 1000 L Flexel 3D Magmix ® bag. Mixing was set to 125 rpm. The cell broth was auto-matically regulated to pH 5 with 36.8 L of acetic acid (2 M) via FlexAct ® automation. A precipitation phase of 45 min was set as minimum time to guarantee a complete precipitation process. During precipitation time 26.5 kg of Diatomaceous Earth were added. The 18 × Sartoclear Dynamics ® Filters were clamped with a tension force of 25 Nm in Sartoclear ® Filter holders.

2.3 Process design for 1000 L CHO HarvestIn contrast to other harvesting technolo-gies Sartoclear Dynamics ® is not affected by viability changes of cell cultures. Because of this unique characteristic surface area and DE requirements are relatively constant. Based on experiences with various mammalian cell cultures a safe amount of Sartoclear Dynamics ® filters and DE can be easily determined based on the cell culture biomass content expressed as the wet cell weight (WCW [g/L]).Furthermore, lowering the process pH to 5.0 has shown to have a great positive influence on filterability, thus reducing the amount of DE required. In low pH conditions, a ballpark figure process layout calculation can be made in the following three steps:

1. Measure your biomass in WCW [g/L]At the harvest day a sample was taken from the bioreactor and centrifuged at 5000 g for 10 min. The determined WCW was 102.5 g/L. For a volume of 1000 L the filtration system should be able to remove a total biomass of 102.5 kg.

Figure 2: Three steps process design for 1000 L CHO Harvest

102.5 g/L

For low pH conditions (~5.0), each Sartoclear Dynamics ® Filter can remove between 6.0 and 6.5 kg biomass

18× Sartoclear Dynamics ® Filters

Use 1.5 kg DE per Sartoclear Dynamics ® Filter

2 × 10 kg Sartoclear Dynamics DE Bag1 × 5 kg Sartoclear Dynamics DE Bag1 × 1.5 kg Sartoclear Dynamics DE Bag

Measure your biomass in WCW [g/L]

Determine your number of Sartoclear Dynamics ® Filters

Determine your quantity of Sartoclear Dynamics ® DE Bags

2. Determine your number of Sartoclear Dynamics ® FiltersFor low pH conditions e.g. pH 5, each Sartoclear Dynamics ® Filter can remove between 6.0 and 6.5 kg biomass. Based on 6.0 kg biomass per filter a quantity of 18 Sartoclear Dynamics ® Filters is required to remove in total 102.5 kg of biomass.

3. Determine your quantity of Sartoclear Dynamics ® DE bagsIt is recommended to use 1.5 kg of DE per Sartoclear Dynamics ® Filter. This results in a total DE consumption of 27.0 kg. As for mammalian cell cultures, it was found out that optimal ratio of DE | WCW lies between 0.25 and 0.30, the total amount is reduced to 26.5 kg.

Table 2: Automation parameters for DBF Filtration

FlexAct ® automation parameters Set valuepH value for pH adjustment 5

Filtration flow 18.9 Lpm

Maximum filtration pressure 21 psi (1.5 bar)

pH value for neutralization 7

Flush volume for recovery 135 L

3 Results and discussion

3.1 Cell CultivationThe CHO fed-batch process was finished on day 17 of cultivation. The cell broth had a VCD of 17.7 × 106 cells/mL and a viability of 81 %. The production of IgG1 peaked on day 17 with a concentration of 8.0 g/L. The cell growth exceeded golden batch growth.

NaOH(1M)

NaOH (1M)

F

P2

Acetic acid(2M)

Acetic acid (2M)

1000L Flexel 3DMagmix® Bag

1000L Flexel 3D Magmix® Bag

WM 520WM 520 WM 520WM 520

WM 701WM 701

18x SartoclearDynamics® Filters18x Sartoclear

Dynamics® Filters

2x Sartopore® 2(0,2µm; 20")

2x Sartopore® 2 (0,2µm; 20")

Sterile connectionvia Biowelder®

Sterile connection via Biowelder®

BioPat®

pressure sensorBioPat®

pressure sensor

BioPat®

ppressure sensorBioPat®

pressure sensor

1000L Flexel 3DMagmix® Bag

1000L Flexel 3D Magmix® Bag

BioProTT™ flow sensor

Sartoclear Dynamics®

DE Bags (26.5kg)Sartoclear Dynamics®

DE Bags (26.5kg)

PBS135LPBS135L

P1

1000L STR®

Bioreactor1000L STR® Bioreactor

WM 720WM 720

Figure 3: Schematic overview of the DBF filtration with 18× Sartoclear Dynamics ® filters

Figure 4: Viable cell density and viability trend

All parts of the set up were connected as shown in Figure 3. The filtration was start-ed with a constant flow rate of 300 LMH, i.e.18.9 Lpm. To avoid any further precipi-tation reaction throughout the filtration process, the filtrate was automatically regulated to pH 7 with NaOH (1 M).

After completion of the filtration the Sartoclear Dynamics ® filters were flushed with 135 L 1× PBS pH 7 buffer (7.5 L per filters) for product recovery. Finally, all filters were drained with compressed air at 5 psi before disassembling.

A remarkable reduction of residual DNA of more than one log compared to the non-clarified material was achieved during harvesting. This early DNA reduction during harvesting contributes to prolong the chro-matography column lifetime and reduces the load on further purification steps.

The overall turbidity was reduced in both runs from 2546 NTU to 16-20 NTU in a single step. This turbidity is adequate for filtration over a sterilizing-grade filter, such as the Sartopore ® 2.

The amount of Sartoclear Dynamics ® Filters and Sartoclear Dynamics ® DE bags used resulted from the direct three steps esti-mation of the required consumables. An optimal setup in terms of consumables would have led to a body feed filtration which can be run at constant flow rate until 21 psi and then at constant pressure until 150 LMH (see User Instructions n° 85037-547-96: “Sartoclear Dynamics ® Filters and Sartoclear ® Depth Filters”). As the filtration finished at 12 psi; further optimization with less DE and less filtration area is possible. For a precise process design, the concentration of DE and the surface area required should be checked at lab scale prior to scaling-up.

In order to facilitate the evaluation of the required lab space at production scale, the following footprint can be taken into account.

A minimum room height of 3.15 m was required to perform transfer of Celpure ® C300 into the Flexel 3D Magmix ® bag. The total footprint needed was 16 m² (here 2 m × 8 m). In comparison to the typical setup using a multi-use centrifuge and depth filtration skids, Dynamic body feed filtration is a compact solution for cell harvesting.

3.2 Dynamic body feed filtrationBesides the full scale setup for clarification of the 1000 L cell broth with 18 × Sartoclear Dynamics ® filters; an additional experiment with a single filter cassette was performed to demonstrate the linear scalability of the technology.

Pressure increase in 1 × and 18 × Sartoclear Dynamics ® Filters is identical (grey and yellow curves), which indicates are linearly scalable. Moreover, linear pressure increase indicates a proper DE consumption.

DBF Filtrations, at process and pilot scale, show an antibody recovery of > 90 %. The investigation indicates that the flush step of Sartoclear Dynamics ® allows a recovery improvement of approx. 12 %, making this step essential for an acceptable recovery.

Figure 5: Comparison of filtration performance between 1 × and 18 × Sartoclear Dynamics ® Filters

Table 3: Detailed results of 1000 L CHO harvest amass balance = filtrate conc.IgG × filtrate volume | starting conc.IgG × starting volume fluid

CHO cell culture WCW = 102.5 g/L; VCD = 17.7 Mio/mL; Viability = 81 %; Turbidity = 2546 NTU;

18 × Sartoclear Dynamics ® (3.78 m²) 1 × Sartoclear Dynamics ® (0.21 m²)

IgG in pretreated cell culture(low pH with DE)

6.2 kg 0.55 kg

Turbidity in clarified cell culture 20.0 NTU 16.1 NTU

IgG (mass balance) before recovery step 77.0 % 78.6 %

IgG (mass balance) Flush volume for recovery 92.0 % 90.0 %

DNA in filtrate 41.5 ppm [-1.2 log] 18.6 ppm [-1.5 log]

Further Readings:– High-Cell-Density Clarification By

Single-Use Diatomaceous Earth Filtration, BioProcess Int. 12(4), 2014, 36-44

– Diatomaceous Earth Filtration: Innovative Single-Use Concepts for Clarification of High-Density Mammalian Cell Cultures, 12(8), 2014, 25-28

– Brochure n° SPK1565-e: Sartoclear Dynamics ®, Single-use Clarification Technology

– User Instructions n° 85037-547-96: “Sartoclear Dynamics ® Filters and Sartoclear ® Depth Filters”

Figure 6: Required footprint for a 1.000L FlexAct ® DBF cell harvest process solution.

4 ConclusionAfter the successful CHO fed-batch culti-vation resulting in a biomass of 102.5 kg, Sartoclear Dynamics ® technology was used for the cell removal step. The amount of DE and the number of devices for collecting the overall biomass were calculated based on the determined wet cell weight. For this amount of biomass, a total number of 18 Sartoclear Dynamics ® Filters and 26.5 kg of DE were used, representing 3.78 m² filter area. High density cell broth containing 6.2 kg IgG was successfully clarified within 4.15 h, including time for flushing and draining to increase recovery. Applying the post-use flush procedure (recovery step) increased the product yield to 90 %.

Linear scalability was demonstrated in an additional run with only one Sartoclear Dynamics ® Filter. Process setups with filter areas from 0.21 m² (one cassette) and 3.78 m² (18 cassettes) showed identical normalized filtration performance.The filter cassettes can be used in different holder sizes, holding up to 36 cassettes (3× stacked rows of 12 cassettes).In conclusion, the Sartoclear Dynamics ® technology proved to be a robust and reliable disposable harvesting technology delivering consistent performance at different scales and resulting in short processing time and high product yield.

8m

1.000 L Palletank

Cubical Mixing1.000 L Palletank

Cubical Mixing

work area

FlexAct® COM Sartoclear Dynamics®

Process Filter Holder

2m

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