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A Rapid, Low-risk Approach for Process Transfer of Biologics from Development to Manufacturing ScaleSebastian Ruhl1, Naomi de Almeida1, Melisa Carpio2, Jens Rupprecht1, Gerhard Greller1, Jens-Christoph Matuszczyk1*

1Sartorius Göttingen, Germany. 2 Sartorius North America. For more information, please contact [email protected]/en/products/fermentation-bioreactors

AbstractManufacturing of biologics commonly focuses on an upstream process with Chinese hamster ovary (CHO) cells in a production bioreactor. Before this can begin, scientists have to find the best culture parameters using shake flasks or mini bioreactors. They then transfer these parameters from the laboratory through different scales of bioreactor. Typically, process transfer during scale-up involves transferring through several different pilot and manufacturing scale bioreactors and doing multiple iterations to optimize process parameters at each scale, all of which is expensive and can take many months to get right. This poster highlights a novel scale conversion tool used for scaling parameters in a bioreactor-size-independent way to predict the best set of cell culture parameters for single-use, stirred tank bioreactors from development through to manufacturing scale. This proof-of-concept study shows it is possible to scale up a cell culture process from mini (15 mL and 250 mL Ambr® bioreactors) to pilot and 2000L (Biostat STR® bioreactors) without affecting the process or product quality.

Materials and Methods- Cell Line – Cellca CHO DG44 expressing a human IgG1 mAb- Bioreactors – Ambr® 15 Cell Culture, Ambr® 250 High Throughput, Univessel® 5L, Biostat STR® 50L, 200L, and 2000L- Process Setpoints – Temperature 36.8°C, pH 7.1, DO 60%- 12-day fed-batch culture with sampling for metabolites, VCC, viability, titer, and N-glycans for all bioreactors- BioPAT® Viamass sensor was used for the Univessel® 5L, Biostat STR® 50L, 200L, and 2000L

Scale Conversion Tool- Bioreactor agitation rates were set according to the scale conversion tool to correlate with a Reynolds number (>3,000), tip speed (0.6-1.25 m/s) and a specific power input of 30 – 200 W/m³ across scales (Table 1)

Figure 2. DO profile (2a top) and pH (2b bottom) across all scales over 12 days

Process Transfer Performance- The DO and pH profiles across all scales are comparable (Figures 2a and 2b)- Temporary decreases in DO for the Ambr® is due to opening of the vessel during automated sampling and feeding- Temporary increases in pH across all scales is due to the addition of a basic feed

Figure 3. VCC and Viability (3a top) and glucose, lactate and osmolality profiles (3b bottom) across all scales over 12 days. Dashed black lines in Figure 3a and 3b represent historical data for each parameter measured +2 and –2 Standard Deviations

Bioprocess Performance at Different Scales- The CHO cell culture showed comparable performance with regards to cell

growth, metabolic profiles and product quantity/quality at all scales- Peak VCD of 20–26 x 106 cells/mL on Day 7-8 with harvest viabilities between 80-90% (Figure 3)- Glucose, lactate, and osmolality profiles consistent throughout the run (Figure 4)- All results comparable to historical golden batch reference data (mean of 30 different Ambr® 250, Univessel® and Biostat STR® bioreactor sizes with CHO cells expressing a commercial IgG)

Scalability of On-Line Analytics- Linear regression capacitance model constructed using online BioPAT® Viamass data measured at a single-frequency indicates transferability between scales during the exponential growth phase with an R2 value of 0.99 (Figure 4)

mAb Titer and CQAs- The daily and total product concentrations showed comparable trends across all scales with harvest concentrations of 2.5-3.5 g/L being achieved on Day 12 (Figure 5)- Critical Quality Attributes of the mAb were not affected during process transfer and showed similar trends in glycosylation patterns of G2/G0 and G1/G0 ratios and comparable binding potencies with between 85-90% fucosylated forms produced from cells cultured from the complete bioreactor range (Figure 5)

Principle Components Analysis- Principle Component Analysis (PCA) was run using SIMCA® Multivariate Data Analysis (MVDA software with VCC, viability, glucose, lactate, osmolality, cell diameter, product quality and final titer data from the Ambr® and Biostat STR® bioreactor runs)- This analysis demonstrates that small scale batch data clusters closely together near to the center of the plot with larger scale bioreactors clustering together on one side of the plot due to their generally higher VCCs and titers- All batch data are within the 95 % confidence region, indicating scalability of the process from 15 mL to 2000 L. (Figure 6)

Figure 4. Linear regression model of capacitance and VCC over 12 days for 5L to 2000L bioreactor scales

ConclusionUtilizing the novel scale conversion tool, it possible to scale up a cell culture process from the process development scale (Ambr® multi-parallel bioreactors and Univessel® benchtop bioreactors) to pilot and commercial manufacturing scale (Biostat STR®) without affecting the process or product quality.

Using this scale-up approach offers bioprocess scientists the potential for developing faster, more cost-effective cell culture processes for their biologics manufacturing

AcknowledgementsWe would like to thank the global Sartorius R&D teams for generating the bioprocess data for this project

We would also like to thank Adrian Stacey at Sartorius Royston for providing the prototype of the scale conversion tool and for his advice on its application and Marek Hoehse at Sartorius Göttingen for generating PCA analysis plots.

Ambr® 15 Cell Culture Ambr® 250 High Throughput

Univessel® Glass 5L

Biostat STR® 50 - 2000L

- Output from the tool shows Reynolds Number (Re), tip speed and P/V changed according to agitation rate, with functions developed with a score from 0 to 1 over the range of the parameters, where 1 represents the optimum for each parameter (Figure 1)

Figure 1. Scaling tool data showing the optimum Re, PPV and tip speed based on the agitation rate

Util

ity [-

]

1

0.8

0.6

0.4

0.2

10,000 30,00020,000

Reynolds number [-]

040,000

Util

ity [-

]

Util

ity [-

]

11

0.80.8

0.60.6

0.40.4

0.20.2

50 100 150 200 250 300 350 400 0.5 1.0 1.5 2.0 2.5 3.0

P/V [W/m3] Tip Speed [m/s]

00

pH [-

}

Time [d]

7.5

7.4

7.3

7.2

7.1

7

6.9

6.80 1 2 3 4 5 6 7 8 9 10 11

D0

[%Sa

t.]

Time [d]

100

90

80

70

60

50

40

30

20

10

00 1 2 3 4 5 6 7 8 9 10 11 12

12

DO Biostat STR® 2000L

DO Ambr® 250


DO Ambr® 15

DO Biostat STR® 50L DO Univessel® 5L


DO Ambr® 250


DO Ambr® 15

DO Biostat STR® 50L DO Univessel® 5L

Bioreactor Reynolds Number Tip Speed (m/s) Specific Power Input W/m³ Agitation Rate (rpm)

Ambr® 15 (15 mL) 3638 0.66 190 1050

Ambr® 250 (250 mL) 13910 1.252 183 855

Univessel® Glass (5L) 38563 1.252 54.1 327

Biostat STR® (50L) 79728 1.252 30.4 162

Biostat STR® (200L) 147427 1.254 30.5 121

Biostat STR® (2000L) 407807 1.258 29.6 70

Table 1. Bioreactor agitation parameters

VCC

[10

6 cells

/mL]

Prod

uct C

once

ntra

tion

[g/L

]

Time [d]

VCC

[10

6 cells

/mL]

Viab

ility

[%]

100

90

80

70

60

50

40

30

20

10

0

25

25

30

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

30

20

20

15

15

10

10

5

5

0

0

1 2 3 4 5 6 7 8 9 10 11 12

Time [d]

0

VCC Historic Data ± 2 SD

Glc Ambr® 15

Glc Historic Data ± 2 SD

VCC Biostat STR® 50L

Prod. Conc. Biostat STR® 50L Prod. Conc. Biostat STR® 200L

Prod. Conc. Biostat STR® 2000L

Glc Biostat STR® 50L

VCC Ambr® 15



VCC Ambr® 250

Glc Ambr® 250



VCC Univessel® 5L

Glc Univessel® 5L

Capacitance Univessel® 5L

Capacitance Biostat STR® 2000L


Linear (All data)


Viability Historic Data ± 2 SD

Viability Ambr® 15

LAC Historic Data ± 2 SD

Osmolality Historic Data ± 2 SD Osmolality Ambr® 15

Osmolality Ambr® 250

Biostat STR® 2000L

Osmolality Univessel® 5L Osmolality Biostat STR® 50L Osmolality Biostat STR® 200L

Viability Biostat STR® 200L

LAC Biostat STR® 50L

Viability Ambr® 250

LAC Ambr® 15


Viability Biostat STR® 200L Viability Biostat STR® 2000L

Viability Univessel® 5L

LAC Ambr® 250


LAC Univessel® 5L

Glu

cose

| La

ctat

e [g

/L]

9

10

8

7

6

5

4

3

2

1

01

10

2

20

3

30

4

40

5

50

6

60

7

70

8 9 10 11 12

Time [d]

Capacitance [pF/cm]

y = 0.3985x + 0.4653R2 = 0.9897

0

0

Osm

olal

ity [m

Osm

ol/k

g]

400

350

300

250

200

150

100

50

0

10 2 3 4 5 6 7 8 9 10 11 12

Prod. Conc. Historic Data ± 2 SD Prod. Conc. Ambr® 15 Prod. Conc. Ambr® 250

Prod. Conc. Univessel® 5L

Figure 5. Daily product concentration (5a top) and cumulative product concentration, glycosylation and fucosylation profiles (Figure 5b bottom) across all scales over 12 days. Dashed black lines in Figure 5a represent historical data +2 and –2 Standard Deviations

G1/GO ratio G2/G0 ratio Fucosylated forms [%] Prod. Concentration [g/L]

Ambr® 15 Ambr® 250 Univessel® 5L Biostat STR® 50L Biostat STR® 200L Biostat STR® 2000L

Rat

io [-

] Pro

duct

Con

cent

ratio

n [g

/L]

Fuco

syla

tion

[%]

4.0

3.5

3.0

2.5

2.0

1.5

100

90

80

70

60

50

40

30

20

10

0

1.0

0.5

0

Figure 6. Batch level model generated on the basis of a PCA of bioprocess data across all scales

t [1]R2x[1] = 0.357, R2x[2] = 0.254, Ellipse: Hotelling’s T2 (95%)

t [2]

-20-25

25

20

15

10

5

0

-5

-10

-15

-20

-25-15 -10 -5 0 5 10 15 20 25

Ambr® 250

Biostat STR® 2000L

Biostat STR® 200L

Ambr® 15

Biostat STR® 50L

Univessel® 5L

a rapid, low-risk approach for process transfer of biologics … · a rapid, low-risk approach for...

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