Download Recombinant LONG®R3IGF-1 and rTransferrin

Company Information
Recombinant LONG®R3IGF‐1 and rTransferrin Improved Growth and Productivity of Two Suspension Chinese Hamster Ovary Cell lines
Repligen Bioprocessing, 41 Seyon Street Building #1, Suite 100 Waltham, MA 02453, www.repligen.com/bioprocessing
Jinghui Qian, Rina Gandhi, Ariane Marolewski
Repligen Corporation, Waltham, MA, USA
LONG®R3IGF‐1 Yielded Significantly Higher Titer than rInsulin in Fed‐batch Culture using CD OptiCHO Medium – Cell Line B
LONG®R3 IGF‐1 is a human IGF‐1 analog containing a 13 amino acid N‐terminal extension and a mutation at position 3. It activates the Type 1 IGF receptor, which is responsible for growth‐promoting and protein synthesis effects in CHO cells. A common growth factor supplement used in CHO media, insulin, acts primarily through the IGF‐R. LONG®R3 IGF‐1 has previously been shown to increase CHO productivity. However, past characterization studies used primarily DMEM/F12 media. As serum‐free media development has progressed, media have become richer and more complex. To establish the effect of LONG®R3 IGF‐1 in media that meet today’s industry standards, the effect of LONG®R3 IGF‐
1 on the productivity of two antibody‐expressing cell lines was compared to that of insulin and no growth factor supplemented media. The studies were carried out in TPP Tubespin 50mL bioreactor tubes. Media with LONG®R3 IGF‐1 gave superior titers versus no growth factor or insulin in several instances. In addition, it was demonstrated that combination of LONG®R3 IGF‐1 and rTransferrin gave higher titer than commercial ITS (rInsulin, rTransferrin, Selenium) solution in several modern commercial media types. LONG®R3 IGF‐1 is used at low, 10 – 100 ng/mL, concentrations in cell culture and is currently used in several commercial antibody manufacturing processes. It represents a regulatory‐friendly method of enhancing serum‐free cell culture performance. LONG®R3 IGF‐1 should, therefore, be considered as a media supplement to further optimize a protein‐free process or to replace insulin in existing processes. • Feed: Efficient Feed A
91%  over no GF
91% no GF
LR3 10 ng/mL
LR3 100 ng/mL
INS 1 µg/mL
INS 10 µg/mL
100
Titer (mg/L)
Summary
LONG®R3IGF‐1 and rInsulin Supplementation Signifciantly Increased Titer in Fed‐batch Culture using CD OptiCHO Medium– Cell Line A
50
0
5
7
Days in Culture
11
• Feed: Efficient Feed A
no GF
LR3 5 ng/mL
LR3 10 ng/mL
LR3 25 ng/mL
LR3 50 ng/mL
LR3 100 ng/mL
INS 0.5 µg/mL
INS 2 µg/mL
INS 5 µg/mL
INS 10 µg/mL
350
300
Titer (mg/L)
250
200
LONG®R3IGF1‐rTransferrin‐Selenium Combination Yielded Significantly Higher Titer than ITS in Fed‐batch Culture using CD FortiCHO Medium
37%  91% over no GF
• Feed: Efficient Feed A
No GF
200
150
ITS (1/100)
83% 91% over ITS
LR3 100 ng/mL, Tf 5 µg/mL
100
LR3 100 ng/mL, Tf 5 µg/mL, Se 6.7 ng/mL
150
Titer (mg/L)
50
0
6
10
Days in Culture
15
LR3 100 ng/mL, Tf 5 µg/mL, Se 0.7 ng/mL
100
50
LONG®R3IGF‐1 Supplementation Signifciantly Increased the Growth and Productivity of Cell Line A in 5‐Liter Bioreactors using CD OptiCHO Medium
8
Days in Culture
10
LONG®R3IGF1 Had Better Stability than rInsulin in CD OptiCHO Medium
91% 43%  over no GF
10
6
120
LR3 (no cells)
8
rInsulin (no cells)
100
LR3 (with cells)
6
4
2
no Growth factor
100 ng/mL LR3
0
0
2
4
6
8
10
Days in Culture
12
14
16
18
% Remaining Growth Factor
Viable cell density (10e6 VC/mL)
• 5L CelliGen BlU bench‐scale bioreactor
• Fed‐batch culture: Medium: OptiCHO; Feed: Efficient Feed A
12
0
rInsulin (with cells)
80
60
40
20
0
0
120
no Growth factor
100 ng/mL LR3
1
2
3
4
5
6
7
8
9
Days in Culture
10
11
12
13
14
15
100
91% 2.7‐fod 
over no GF
Titer (mg/L)
80
Conclusions
• Application data for LONG®R3IGF1 and rTransferrin were generated using recombinant CHO cell lines and modern commercial chemically‐defined, Animal‐Derived‐Component free media and feeds
60
• LONG®R3IGF1 is more effectivet than rInsulin in increasing protein synthesis from recombinant CHO cell lines when supplemented to growth factor free media
40
• LONG®R3IGF1‐rTransferrin combination is more potent than rInsulin‐rTranferrin‐Selenium solution in increasing the productivity of recombinant CHO cell lines
20
• LONG®R3IGF1 yielded higher or comparable titer to rInsulin when used at 1000‐fold lower concentration . It is therefore a very powerful mitogenic growth factor that is economical to use
0
0
2
4
6
8
10
Days in Culture
12
14
16
18
• LONG®R3IGF1is produced in a validated, cGMP (ICH Q7) compliant, manufacturing process with no animal derived components; Final product is released by QC testing
• Ask us about other applications of our growth factors [email protected]
• CHO culture: cell line development, cell banking
• Stem cell culture and regenerative medicine