comparison between disposable and conventional
TRANSCRIPT
Comparison between disposable and conventional chromatography in purification
of r-h FSH M. Fahim1, F. Mueller1, J. Rohde1
1Rhein-Minapharm Biogenetics, R&D, Mina Street, 3rd Industrial Zone A2, 10th of Ramadan City, Egypt,
The use of disposables in downstream processes is well established in terms of different filtration technologies, buffer preparation and storage of liquids and became increasingly popular over the last years. Disposable systems are generally known to have several significant advantages in comparison to conventional concepts such as risk reduction for cross contamination. Disposables also save costs, reduce buffer consumption [1], labor and capital space and minimize time of cleaning and cleaning validation of approximately 30% [2]. Recently, a number of new technologies for single use chromatography systems attracted many applicants to integrate them in downstream process strategies [3]. We explored disposable chromatography as a capture and/or intermediate step [Sartobind® Q and Sartobind® Phenyl] [4] in a defined purification process of r-h FSH. We herein compare the conventional chromatography pathway (P1) and alternative pathways which include one or two disposable chromatography steps (P2 and P3 ) in respect to their elution profile, yield and purity of the target protein. Results were found to be comparable with several advantages of the disposable membrane
adsorber, in addition to the above mentioned benefits; the disposable membrane adsorber solved a carryover problem monitored with the traditional chromatography in the capture step.
Process flow chartr-h FSH in
ultra filteredsupernatant
Path a 2 3
Capture step (IEC)
Intermediate step (HIC ‐ Phenyl)
Polishing step(HIC‐ Octyl)
Pathway
1Pathway
2Pathway
3Pathway
1Pathway
2Pathway
3Pathway
1Pathway
2Pathway
3Q Sartobind Sartobind Phenyl Phenyl Sartobind
1- RP-HPLCResults and discussion
Pathway 1Conventional
columns
(1) Q-Sepharose
Y= 60%
(2)
Pathway 2One
disposable step
(1) Sartobind Q
Y= 72%
(2)
Pathway 3Two
disosablesteps
(1) Sartobind Q
Y=73%
(2) Sartobind
ColumnQ‐
SepharoseSartobind
QSartobind
QPhenyl HP
Phenyl HP
Sartobind Phenyl
Octyl Octyl Octyl
FSH loaded [mg] 6.25 8.63 8.63 3.13 4.24 1.96 1.04 1.3 0.99
FSH loaded per ml resin [mg/mL]
1.25 1.23 1.23 0.62 0.84 0.65 0.20 0.26 0.19
Eluted FSH [mg] 3.81 6.24 6.30 1.10 1.68 0.96 0.67 0.84 0.58Yield [%] 60.96 72.30 73.00 35.14 39.62 48.97 64.42 64.61 58.58
Recovery [%] 84.32 88.99 92.46 78.91 100.00 81.63 91.34 100.00 88.88
Comments
Comparable elution profiles with higher yield for the disposable
column ( Sartobind Q) in
Different elution steps with different profiles due to early
elution in case of the disposable column (Sartobind
Comparable profiles and yields
Phenyl HPY= 35%
(3) Octyl
Y= 64%
Overall yield13%
Phenyl HPY= 39%
(3) Octyl
Y= 64%
Overall yield18%
SartobindPhenylY= 48%
(3) Octyl
Y= 58%
Overall yield20%
( )comparison to the conventional
one (Q‐Sepharose)
p (Phenyl) but with higher yield
in comparison to the conventional one (Phenyl HP)
2- SDS-PAGE & Western blot
KDa
116.397.466.3 55.4
Table1 presents overview of the RP-HPLC analysis results of the three different pathways (P1, P2 and P3).
r‐h FSH before purification r‐h FSH after purification
Intact purified FSH
P1 P2 P3 St.KDa
116.397.466.3 55.4
P1 P2 P3 St.
Lane Sample Name Lane Sample Name1 Marker 2 Supernatant3 Purified FSH (Pathway1) 150IU 4 Purified FSH (Pathway1) 75IU5 Purified FSH (Pathway2) 150IU 6 Purified FSH (Pathway2) 75IU7 Purified FSH (Pathway3) 150IU 8 Purified FSH (Pathway3) 75IU
Figure 2 presents Silver stained SDS-PAGE and Western Blot of supernatant containing r-h FSH before and after purification by the three different pathways (P1, P2 and P3). Main fraction containing purified r-h FSH diluted to 150 and 75 IU in comparison to
standard with the same concentration.Materials: Q-Sepharose FF column; CV= 5mL, Phenyl HP ® column; CV= 5mL, Sartobind® Q membrane; CV= 7mL, Sartobind ® Phenyl membrane; CV= 3mL, Octyl FF ® column; CV= 5mL, ÄKTA explorer 100.
Materials and Methods
36.5 31.5
21.5
14.4
Figure1 presents flow chart of the three different purification pathways (P1, P2 and P3); Y= Yield.
1 2 1 3 4 5 6 7 8 910 1 3 4 5 6 7 8 910
Degraded FSH
36.5 31.5
21.5
14.4Remaining impurity
Conclusions and outlook9 Standard r-h FSH 150IU 10 Standard r-h FSH 75IU
Disposable chromatography systems have several advantages in comparison to conventional columns. They minimize time of cleaning and cleaning validation and solve the carry over problem specially in the capture step. That encouraged us to apply it in different steps in the purification process of r-h FSH which is under development.
Application of one disposable chromatography step (Sartobind Q) in the capture step of r-h FSH purification process was comparable to the conventional column (Q-sepharose) regarding the elution profiles and purity. An additional advantage
i i th i ld b 10% f thi t d 5% f th ll i ld
Methods:Ultra filtered CHO culture supernatant containing r-h FSH was purified as follows:
[P1]: Application of conventional columns: Capturing (Q-Sepharose), intermediate purification (Phenyl HP) and polishing(Octyl FF) of r-h FSH. 1.25, 0.62 and 0.2 mg FSH were loaded per[ml] resin on the three different columns.
[P2]: Application of one disposable chromatography capture stepusing Sartobind Q with subsequent continuation of purification byconventional columns (Phenyl HP and Octyl). 1.23, 0.84 and 0.26mg FSH were loaded per [ml] resin.
References:[1] Govind Rao et al. Non Invasive Sensors as Enablers of “Smart” Disposables. BioProcess International. 2009; vol.7, supplement1[2] D'Aquino, Rita: Bioprocessing Systems Go Disposable. Chemical Engineering Progress May 2006.[3] Disposables Improve Downstream Process. Euro Bio Tech News.2008; vol.7,7-8[4] Stefan Fischer. Sartobind Membrane Adsorbers. August 2009 (www.sartorius.com).
was an increase in the yield by 10% for this step and 5% for the overall yield.
Application of two disposable chromatography steps (Sarobind Q and SarobindPhenyl) in the capture and intermediate step of the r-h FSH purification processshowed a minimal decrease in final product purity but the overall yield increasedby 7%. Further development for the Sartobind phenyl step is still needed toimprove the final product purity and maintain the high yield. Future applicationsin different pipeline processes are currently under evaluation.
g p [ ][P3]: Involved the application of two disposable chromatography
steps (capture and intermediate) using Sartobind Q and SartobindPhenyl, followed by conventional polishing (Octyl FF).1.23, 0.65 and0.19 mg FSH were loaded per ml resin.Flow rate: 5 mL/min. All fractions eluted were analyzed by RP-HPLCand silver stained SDS-PAGE as well as Western Blot.