Peer-riewed:single-Use Bioreactors single-Use Bioreactors …€¦ · · 2015-11-23The article...

22 BioPharm International www.biopharminternational.com October 2014

In the current competitive bio-

pharmaceutical production land-

scape, the substantially higher

proce s s deve lopment cos t s

mean that new candidate molecules

are increasingly under pressure to

advance to clinical proof-of-concept

stage in the shortest possible time with

a minimum commitment of capital

resources. Single-use bioreactors have

the potential to alleviate both of these

constraints because they confer spe-

cific advantages over conventional

stainless-steel bioreactors. Acquisition

and implementation costs of dispos-

able bioreactors tend to be much lower

compared to traditional stainless-steel

bioreactor systems. Some of these

advantages can be attributed to their

modular nature, which enables the

use of existing manufacturing facili-

ties with minimal modifications to the

current infrastructure, allowing execu-

tion of a straightforward “roll in-roll

out” concept. Furthermore, because

the product contact surface is changed

with each experiment/campaign, carry

over between fermentations is non-

existent, thus removing the need to

perform cleaning verification/valida-

tion and enabling a more rapid turn-

around between products. As a result,

single-use systems from various manu-

facturers are now available on the mar-

ket, and pharmaceutical development

groups have increased the frequency

with which these systems are used to

produce biopharmaceuticals (1, 2).

Although the benefits concerning

this technology are fairly clear, one

of the primary unresolved concerns

focuses on performance comparabil-

ity between single-use and conven-

tional stainless-steel systems. In this

study, the authors present data from

two different fed-batch processes pro-

ducing monoclonal antibodies (mAbs)

using both system types. To achieve

good correlations between bench-scale

development reactors and the single-

ABSTRACT

The article describes successful incorporation of single-use bioreactors as part of

a fed-batch platform technology for the production of clinical biopharmaceuticals.

By matching general reactor characteristics, the authors were able to scale-up

fed-batch processes from traditional bench-scale bioreactors to the large-scale

single-use systems without any significant operational differences or changes

in critical product quality attributes.

single-Use Bioreactors for the rapid Production of Preclinical and clinical

BiopharmaceuticalsRüdiger Heidemann, Christopher R. Cruz, Paul Wu, Mikal Sherman, Jessica Martin, and Christel Fenge

Rüdiger Heidemann, PhD, is senior staff

development scientist, Christopher R. Cruz

is senior associate development scientist,

and Paul Wu, PhD, is director, Upstream

development, all three at cell culture

development, Global Biological development,

Bayer healthcare llc, Berkeley, ca 94701;

Mikal Sherman is application specialist,

fermentation technologies, and Jessica Martin is field marketing manager, single-Use

Bioreactors, both at sartorius stedim north

america, Bohemia, nY 11716, Usa; and Christel Fenge, PhD, is vice-president of marketing for

fermentation technologies at sartorius stedim

Biotech, 37079 Göttingen, Germany.

PEER-REVIEWED

article submitted: april 14, 2014.

article accepted: august 29, 2014.

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Peer-reviewed: single-Use Bioreactors

ES513390_BP1014_022.pgs 10.03.2014 23:07 ADV blackyellowmagentacyan

October 2014 www.biopharminternational.com BioPharm International 23

use bioreactors, a continuous stirred tank

reactors (CSTR) design was used through-

out the study as described by De Wilde et

al. (3). One major outcome of this work was

the development of an entirely single-use

upstream platform for the manufacture of

biopharmaceuticals from vial thaw to har-

vest clarification.

Materials and MethodsRecombinant Chinese hamster ovary (CHO)

cell lines were used to produce two different

fully human mAbs in fed-batch processes.

Process A was used to produce an IgG2 and

Process B to produce an IgG1. Both processes

used a combination of commercially avail-

able “off the shelf” and proprietary basal

media and feed solutions.

Initial process development was per-

formed in traditional bioreactor designs at

the 2-L, 5-L, and/or 10-L scale (Applikon

Biotechnology, Netherlands). Preclinical and

clinical material was generated in the single-

use Biostat STR (Sartorius Biotech, Germany)

at the 200-L and 1000-L scales. A standard

bag design consisting of two marine-type

impellers, a macro-sparger and optical sen-

sors (PreSens, Germany) for pH and dissolved

oxygen was used. Stirrer geometry, tip speed,

and overall power input were matched as

closely as possible among all bioreactors,

similar to the approach taken by De Wilde

and Adams (4) and Noack et al. (5). Figure 1

illustrates tip speed and power input values

among the different bioreactors. Although

the power input of the 10-L system is slightly

higher than the rest, the overall tip speed

remains comparable to that of the disposable

units.

Standard shaker flasks were used during

the seed train expansion process. Cells were

cultivated in a CO2 incubator maintained at

37.0 oC and 5–7% CO2. Scale-up passaging

occurred every two to three days until suf-

ficient cell mass had accumulated to directly

inoculate the bioreactors regardless of scale,

with the exception of the 1000 L. The 200-L

bioreactor was inoculated at an initial vol-

ume of 50–80 L operation range due to varia-

tion in the accumulated cell mass of the seed

train. Basal medium was added to the reactor

to “passage” the cells by dilution and consid-

ered in “scale-up” mode until a predefined

total cell number was reached; this time

point was defined as fed-batch day zero. If

required, the 200-L unit was used as part of

the seed train expansion process and culture

was directly transferred to the 1000-L reactor

to achieve a target starting cell density on

inoculation day.

Fermenter process parameters (e.g., pH,

temperature, pO2) were maintained by bio-

controllers manufactured by either Sartorius

(DCU 2/3/Biostat STR, Sartorius Biotech,

Germany) or Applikon (iControl, Applikon

Biotechnology, Netherlands). All bioreactors

were configured with a macro-sparger to

deliver mixed gas consisting of air, N2, CO2,

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS


Figure 1: Tip speed and power input values for different bioreactors. Two different types of

impellers were used in the 2 L reactors.

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

25

20

15

10

5

0

Tip speed = Nidπ

Power input =nNpρ Ni

3d5

V

Ni - agitation rate

Np - impeller power number

n - number of impellers

ρ - fuid density

d - impeller diameter

V - reactor volume

2L a 2L b 5L 10L

Tip

sp

eed

(m/ s

)

Po

wer

inp

ut

(W/ m

3)

200L

SUB

1000L

SUB

Tip speed and power input

Contin. on page 26



and O2. Dissolved oxygen was controlled at

40% air saturation, pH at 6.7–7.3, and the

initial process temperature was set to 36.5 oC.

For Process B, a temperature shift to 33.0 oC

was performed once the cell density reached

8 x 106 cells/mL.

The bioreactors were sampled daily to

determine viable cell density and viabil-

ity (Cedex cell counter, Roche Diagnostics,

Germany) as well as glucose and lactate val-

ues (YSI, Yellow Springs Instruments, OH,

USA). Dissolved oxygen, pH, and pCO2 lev-

els were measured offline using a blood gas

analyzer (Siemens Diagnostics, NJ, USA) and

used to verify the pH and dissolved oxy-

gen probes of the bioreactors. mAb titers

were measured using a protein A high-per-

formance liquid chromatography (HPLC)

method. The fed-batch process was termi-

nated after reaching predefined harvest cri-

teria (viability and/or time based), and the

crude harvest was clarified by dead-end fil-

tration.

The 200-L Biostat, in addition to being

the seed source for the 1000-L system, was

also used to verify the bench-scale fed-batch

process and to produce preclinical material.

The 1000-L reactor was used to produce GMP

material for clinical trials. The entire clinical

upstream process consisting of the 200-L and

1000-L bioreactors as depicted in Figure 2,

uses disposable materials throughout, includ-

ing bags for basal media, feeds, and clarified

harvest.

resUlts and discUssionTo compare the performance of the single-

use bioreactors to traditional fermenters, crit-

ical process parameters for each vessel type

are plotted together. Data from multi-use

reactors are plotted as grey dots to define a

point cloud of expected values and single-

use disposable runs are shown as continuous

lines. Figure 3 and Figure 4 summarize the

data obtained in Process A and B, respec-

tively. As shown for Process A, cell growth,

viability, glucose, and lactate concentra-

tions obtained with the single-use systems

follow the general trends defined by the

bench-scale reusable bioreactors. Lactate val-

ues were slightly higher in the bench-scale

reactor cultures. These cultures were used

for the initial process development, specifi-

cally to optimize the feeding process. The

slightly higher viability can be attributed to

slight differences in the Cedex cell counter

used during these cultures. Offline pH and

pO2 measurements are well aligned, dem-


Figure 2: Schematic overview of the upstream fed-batch process using disposable bioreactors.

Duration ~ 14 days

• 1mL cryo-vial• shaker �asks (10L required for inoculation)

Seed-Train

Expansion

200L

BIOSTAT

STR

1000L

BIOSTAT

STR

Cell Sep:

Dead-End

Filtration

Clari3ed

Harvest

Clari3ed

Harvest

• Single-Use / Disposable 3lters already implemented for downstream processes

• 50L min working volume in 200L• 250L min working volume in 1000L

Duration ~ 25 days Duration ~ 1 day

Contin. from page 23



onstrating that single-use bioreactor con-

trol is comparable and was not affected by

vessel type or the measurement technology

(i.e., conventional glass electrodes and Clark

oxygen probes vs. single-use fluorescence

patches). As a result, most Biostat STR data


Figure 3: Process A parameters. Grey squares: bench-scale data (5 and 10 L), blue lines: 200 L

Biostat STR, green lines: 1000 L Biostat STR.

30

25

20

15

10

5

0

8

7

6

5

4

3

2

1

0

7.5

7.4

7.3

7.2

7.1

7.0

6.9

6.8

6.7

6.6

6.5

100

90

80

70

60

50

40

30

20

10

0

140

120

100

80

60

40

20

0

2.5

2.0

1.5

1.0

0.5

0.0

110

100

90

80

70

60

0 2 4 6 8 10 12 14 16

VC

D (

10

6 c

ell

s / mL)

Glu

cose

(g

/L)

pH

pC

O2(m

mH

g)

pO

2(m

mH

g)

Tit

er

(re

lati

ve

un

its)

Via

bil

ity

(%

)

Time (days)

0 2 4 6 8 10 12 14 16

Time (days)

0 2 4 6 8 10 12 14 16

Time (days)

0 2 4 6 8 10 12 14 16

Time (days)0 2 4 6 8 10 12 14 16

Time (days)

offine pO2

pCO2

Titer

offine pH

0 2 4 6 8 10 12 14 16

Time (days)

0 2 4 6 8 10 12 14 16

Time (days)

0 2 4 6 8 10 12 14 16

Time (days)

Viable cell density

Glucose

Cell viability

LactateLa

cta

te (

g/ L

)

Contin. on page 30



were highly comparable to the data obtained

at small scale, and certain parameters shifted

towards more desirable profiles (i.e., reduced

lactate concentrations and increased titer

per unit time). Process B in Figure 4 shows

similar behavior, with tighter clustering of


Figure 4: Process B parameters. Grey squares: bench-scale data (5 and 10 L), blue lines: 200 L

Biostat STR, green lines: 1000 L Biostat STR.

14

12

10

8

6

4

2

0

0 2 4 6 8 10 12 14

VC

D (

10

6 c

ell

s / mL)

Lact

ate

(g/ L

)

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

0 2 4 6 8 10 12 14

Time (days)

Viable cell density110

100

90

80

70

60

Via

bilit

y (

%)

Cell viability

8

7

6

5

4

3

2

1

0

Glu

cose

(g

/L)

Glucose 3.0

2.5

2.0

1.5

1.0

0.5

0.0

Lactate

7.5

7.4

7.3

7.2

7.1

7.0

6.9

6.8

6.7

6.6

6.5

pH

offine pH 140

120

100

80

60

40

20

0

pC

O2(m

mH

g)

pCO2

100

90

80

70

60

50

40

30

20

10

0

pO

2(m

mH

g)

offine pO2

Tit

er

(rela

tive u

nit

s)

Titer




data among the reactor types and scales.

For some of the bench-scale reactors as well

as the 1000-L disposable unit the tempera-

ture shift was programed into the control

unit at a rate of 0.5 oC per 30 minutes. The

temperature decline was linear over the 3.5

h time span with less than 0.5 oC under

shoot. The 200-L disposable unit used a heat-

ing blanket, therefore, active cooling like in

the 1000-L unit was not possible. The tem-

perature shift here took approximately 11 h

with a slightly larger under shoot (data not

shown). Nevertheless, this second process

also demonstrates the feasibility of imple-

menting temperature shift control schemes

with single-use bioreactors. Overall, the tem-

perature shift for Process B was necessary to

obtain the correct product quality attributes.

In addition, it increased the space-time pro-

ductivity of the antibody.

In addition to similar in-process culture

performance, critical product quality attri-

butes of the harvest like protein aggregation

rates, charge heterogeneity, and glycosyl-

ation profiles remained consistent across

all bioreactor systems (data not shown).

Overall, the data show good comparability

between the bench-scale cultures and the

large-scale fermentations, demonstrating

both the scalability of each processes and

the successful integration of single-use bio-

reactor technology into the authors’ clinical

manufacturing platform for mAb fed-batch

processes.

One of the primary concerns surround-

ing the adoption of single-use reactor tech-

nology for full commercial manufacturing

is bag robustness. Due to the nature of the

materials employed, defects can poten-

tially be introduced at any point during the

life of the bag; at manufacture, shipping,

unpacking/setup, or operation. During

the preliminary implementation at Bayer

HealthCare, a 200-L culture was terminated

one day after inoculation because a 24 h

medium sterility-hold at 50 L did not reveal

a pinhole defect. That incident triggered

an in-house integrity test of all single-use

reactors prior to use which consisted of

pressurizing a mounted bag to 0.25–0.35

psi (17–24 mbar) and monitoring the pres-

sure for 10 min (200 L) or 20 min (1000

L), shown in Figure 5. An intact bag will

only lose ~0.02 psi (1.4 mbar) during the

test period whereas a defective bag will rap-

idly depressurize during the test procedure.

Unfortunately, this practice does not guar-

antee success, as the metal wall of the bag


Figure 5: Pressure drop profles for all STR bags. A pressure profle from a defective bag is

shown for comparison.

0.2

0.18

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

0 5 10 15

200 L bag

1000 L bag

defective bag

20

Pre

ssu

re d

rop

(p

si)

Time (min)

Contin. on page 34




holder can mask potential defects during

the pressure hold and despite a passing test.

Bag failure can result in contamination and

lost cultures.

To mitigate this risk, Sartorius Stedim

Biotech developed and qualified a pressure

test comparable to the one described by the

authors but utilizing a patented fleece that

inserts a mesh gap between the bag and

holder surfaces to prevent these potential

masking effects. The approach allows post

bag installation and pre-use testing of the

entire single-use bioreactor assembly (6). The

fleece is designed to be easily removed prior

to the start of a run, to ensure normal bio-

reactor temperature control. This method

has been qualified for reliable defect detec-

tion for various bag sizes (i.e., a correlation

between reliably detectable defect size and

pressure decay has been established) (7).

This method is, therefore, a more reliable

detection method and fully encompasses

any typical defects that might be incurred

during transportation, storage, unpacking,

and installation that cannot be identified

by simple visual inspection. In essence, this

approach is similar to conventional stainless-

steel bioreactor practices, where the steriliza-

tion method is qualified during installation

qualification/operation qualification (IQ/

OQ) and a pressure hold test is often per-

formed for risk mitigation purposes to

detect any miss-assemblies during regular

operation. The sterilization IQ/OQ can be

compared to the vendor assembly and ster-

ilization qualification of single-use bags and

the pressure decay testing serves in a compa-

rable way as a risk mitigation tool.

conclUsionAs more and more single-use bioreactors and

bags are used in late-phase and commercial

production, especially with the availabil-

ity of 2000-L single-use stirred tank biore-

actors, the need for consistent bag quality,

robustness, improved assurance of supply,

change management, and business continu-

ity planning become crucial. To satisfy these

requirements, Sartorius Stedim Biotech

developed a new polyethylene film in close

collaboration with resin and film suppli-

ers to meet future industry needs and to

further improve bag consistency, robust-

ness, and performance for single-use biopro-

cessing applications. During development,

attention was paid to working with ven-

dors to ensure a stable supply chain, clearly

defining and controlling the resin and addi-

tive packages, establishing acceptable film

extrusion ranges with design space studies,

and optimizing of the bag welding process.

The result was a system with significantly

improved strength and flexibility charac-

teristics, alleviating many of the potential

avenues to introduce defects during the

manufacture and handling of these single-

use bioreactors (8). Furthermore, cell culture

and leachable studies were performed during

all stages of the new film development to

ensure the new formulation is not toxic and

does not impede cell growth (9).

All these aspects help to pave the way

towards wide implementation of commercial

scale single-use biomanufacturing, benefit-

ting from the initially mentioned advantages

of reduced upfront investment, flexibility,

quick change-over, and minimal validation

effort.

references 1. C. Heath and R. Kiss, Biotechnol. Prog. 23,

46–51 (2007).

2. M. George et al., Biotechnol. Bioeng. 106, 609–

917 (2010).

3. D. De Wilde et al., BioProcess Int. 7 (Suppl 4)

36–41 (2009).

4. D. De Wilde and T. Adams, Eur. J. Parent. Pharm.

Sci. 15, 41–46 (2010).

5. U. Noack et al., “Single-Use Stirred Tank

Reactor BIOSTAT CultiBag STR: Characterization

and Applications,” in Single-Use Technology in

Biopharmaceutical Manufacture (Eds. R. Eibl and

D. Eibl), John Wiley & Sons, Inc. (Hoboken, NJ,

USA, 2010).

6. M. Stering et al., Genetic Engineering &

Biotechnology News, 33 (11) (2013).

7. M. Stering et al., BioProcess Int. 12 (Suppl 5)

58–61 (2014).

8. E. Vachette et al., BioProcess Int. 12 (Suppl 5)

38–42 (2014).

9. E. Jurkiewicz et al., Verification of a new

biocompatible single-use film formulation

with optimized additive content for multiple

bioprocess applications. Biotechn. Prog., Epub

ahead of print, accepted: 21 May 2014, DOI:

10.1002/btpr.1934. ◆



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