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Ultra Scale-Down Discovery of Low Shear Stress Processing For Selective
Recovery of Next Generation Fusion Proteins
Department of Biochemical Engineering, University College London: E. Lau (presenting author, e-mail: [email protected]), S. Kong, M. Hoare
ImmunoBiology Limited, Cambridge, UK: S. McNulty, C. Entwisle, A. McIlgorm, K. Dalton
Abstract
Fusion protein products potentially pose increased challenges in preparation and purification. Of particular concerns are: (i) the impact of shear stress related effects on product integrity and (ii) the presence of product-related
contaminants which could prove challenging to remove during the high resolution purification steps. USD studies demonstrated the value of low shear stress centrifugation or filtration in achieving selective recovery of the required
molecular form of the product. USD studies were also used to predict improved removal of contaminants such as lipids and nucleic acids and cellular debris when using filtration. These USD predictions were verified at pilot scale.
Introduction
Fc-fusion proteins are molecules in which the immunoglobulin Fc region is fused to a protein of interest (Huang, 2009). One potential application of Fc-fusion proteins is as a vaccine. In this presentation, the processing of a Fcfusion protein vaccine candidate against the Hepatitis C virus (HCV) is presented. Generic processing platforms are in existence for the production of therapeutic grade antibody and antibody-like molecules in order to accelerate the
process development of potential drug candidates. One advantage of the presence of a hIgG1-Fc region is that it enables purification using Protein A chromatography (Huang, 2009) once a suitable level of broth clarification is
achieved using clarification platforms such as centrifugation (Hutchinson et al. 2006; Kelley et al. 2009) or depth filtration (Prashad and Tarrach, 2006; Yigzaw et al. 2006).
Glossary
Equations
 OD s − OD0 
 × 100%
S =
 OD − OD 
f
0 

1) % solid remaining
t
1  1
=
+ *
V * Q0*  Vmax
2) Pore restriction model
3) Pore restriction model
accounted for start-up errors
(t − t0 )
(V
*
− V0
*
) = (V
*

t

1
(t − t0 ) + 1 *
*
− V0
Q0
)
max
Product profile during fermentation
Day
A
MW 4 6 7 8 10 12
188 kDa
98 kDa
98 kDa
% solid remaining
OD600 of sample
OD600 of well spun sample
OD600 of feed
Filtration time (s)
Time for start-up condition (s)
USD prediction of centrifuge capacity (Q/∑) (m s-1)
Total filtrate per filter area (L m-2)
Total filtrate per area for the start-up phase (L m-2)
Min. flux (L m-2 h-1)
Ultra scale-down
S
ODs
OD0
ODf
t
t0
V/t∑
V*
V0*
Q*min
USD
Pilot scale
5 L STR
fermentation
Initial flux post-start-up (LMH)
Fully glycosylated fusion protein with binding
efficacy to human CD64 receptor
Alternative glycosylation form of mIB7
Dimer form of mIB7
Dimer form of mIB7*
Monomer form of mIb7
Monomer form of mIB7*
Q0
mIB7
mIB7*
mIB7d
mIB7d*
mIB7m
mIB7m*
Ultra Scale-Down (USD) approach
mIB7d*
Non-reducing
MW
4 6
B
Day
7 8 10 12
mIB7m
mIB7m*
38 kDa
Reducing
Western blots of bioreactor samples (Human-Fc
detection). Day-4, 6, 7, 8, 10 and 12 samples are
presented. Clarified cell culture samples were purified
using Protein A. Two gel systems were utilised for
Western blot analysis. (A) non-reducing and (B)
reducing. mIB7d* is a different glycosylated variant with
lower molecular weight than mIB7d. In order to reduce
heterogeneity in process stream, the cell culture broth
was harvested in day 6 in order to maximise recovery
mIB7d relative to mIB7d*.
Disc stack continuous
centrifuge
(CSA-1,
Westfalia) with 0.6L
bowl volume
High-speed rotating disc shear
device provides shearing
Cuno ZetaPlus 05SP
depth filter with 25
cm2 filter area
Custom built filter media
housing with 0.28 cm2
• Product-related
contaminant: mIB7*
V* (L m-2)
100
Filtration time (s)
150
200
% Solids
remaining
D
Left: Effect of centrifugation and filtration on Fc-fusion
protein mIB7 recovery and contaminants level.
(A) The recovery levels of ELISA+ve material are
marginally lower in filtration samples.
(B) DNA and (C) Lipid levels in depth filtration are
~50% and ~40% lower than obtained for centrifugation
respectively.
(D) The % solid remaining in USD and pilot-scale depth
filtration experiments are ~60% lower than those
obtained for both centrifugation scales
2
(Note, the final broth load per filter area was similar for
both filtration scales studied, i.e. 1.7 mL cm-2).
1
B
io
n
ifu
ga
tio
n
ga
t
ri
fu
nt
r
d
d
ar
ar
nd
nd
ta n
r sta ion
n
n
er s tio
io
k e 7 at
tio
rk Ib7 ga
at
a
a
ar Ib ifug
n n ug
m m rifu
n n ug
t m d m tr
tio tio trif
ht ied ent
tio atio trif
a
a
g
gh ifie cen
a
n
n
r
i
i
r
lt g e
lt g e
e rif c
e r
w pu ale tion e fi rifu e c
w pu ale tion e fi rifu e c
l t l
c a al nt al
c a
ar
ar
c e c
ul n A s iltr c a en c a
ul n A s iltr
ec ei ch f t-s c t-s
ec ei ch f t-s c t-s
ol rot en SD ilo SD ilo
ol rot en SD ilo SD ilo
M P B U
M P B U
P U P
P U P
sc
al
e
ce
nt
n
at
io
n
ce
P
ilo
t-
ilo
P
U
tsc
al
e
fil
tr
tio
tio
fil
tr
a
ifu
ga
D
S
ce
nt
r
le
SD
n
0
191 kDa
Day 4 USD prediction
Day 6 USD prediction
, Day 4 pilot
, Day 6 pilot
Predictions from pilot data and model
mIB7dimer
mIB7dimer*
mIB7monomer
mIB7monomer*
97 kDa
64 kDa
20
Non-reducing
Reducing
51 kDa
Above: Western blots showing supernatant and filtrate qualities at USD and pilot scale. The filtrates
obtained from both USD and pilot scale are of comparable quality in terms of product molecular weight
bands. They are also similar to those obtained for bench scale centrifugation (no shear) or low shear
stress USD and pilot-scale centrifugation.
0
50
ELISA positive
0.0
3
10
0
0.5
70
(B) % solids remaining in supernatant based on OD600 measurements following centrifugal clarification at various
equivalent V/t∑lab. Low shear treatment mimics hydro-hermetic feed zone found in CSA-1. Significant increases from
30% to 150% in solids carryover are observed as a result of exposure to low shear stress levels in USD studies.
Day 4 USD
Day 6 USD
1.0
-1
Option 2: Depth filtration
A
C
U
60
(A) Western blot of a reducing gel showing sensitivity to shear stress of day 4 samples at (V/t∑)lab of 21.9, 43.4 and
63.7 ×10-9 m s-1. Cells were subjected to no shear stress, low shear stress (0.019×106 W kg-1) and high shear stress
(0.37×106 W kg-1) environments. Lane F is the well spun sample subjected to no shear stress
,
,
materials (mg L-1)
Lipid (g L-1)
50
-9
10
1
sc
a
40
V/tΣ
Σlab (x10 m s )
30
B
en
ch
30
Study of supernatant qualities observed during primary clarification of day 4 and day 6 CHO-S cultures.
30
0
2
0
1
20
40
A
6
DNA
(mg L-1)
2
0
40
12
B
% Solids remaining
B
Day 6 no shear
Day 6 low shear
Day 6 CSA-1
3
Tecan Freedom EVO® robotic
liquid handling platform and data
logging
USD vs Pilot scale
4
20
96 deep square well plates provide
high throughput V/tƉ screen using
bench top centrifuge
• Product: mIB7 (expressed
as dimer)
Option 1: Centrifugation
V* (L m-2)
*
mIB7d
62 kDa
0
Max. volume filtered per filter area (L m-2)
V*max
0
50
100
150
Conclusion
200
Filtration time (s)
Effect of cell culture age on filter performance for USD and pilot-scale constant pressure depth filtration (at 100 mBar).
(A) Raw data for day 4 and day 6 USD filtration.
(B) Comparison of pilot-scale filtration profiles and prediction out of USD analysis. Pilot-scale data is modelled with
resultant V*max values of 40 L m-2 (day 4) and 28 L m-2 (day 6). These compare with predicted values from USD
analysis of 45 L m-2 (day 4) and 24 L m-2 (day 6).
Data obtained from USD and pilot scale processes were comparable in terms of % solid carryover,
product recovery, contaminating DNA and lipids and mIB7d quality. Product variations increased with
culture age. Therefore, to recover a secreted product with minimal product-related variants, using a day 6
harvest combined with depth filtration as primary recovery step is recommended. In conclusion, the USD
techniques employed in this study were predictive of their pilot scale counterpart.
Publication: Eduardo Lau, Simyee Kong, Shaun McNulty, Claire Entwisle, Ann Mcilgorm, Kate Dalton, Mike Hoare, 2013, “An Ultra Scale-Down Characterization of Low Shear Stress Primary
Recovery Stages to Enhance Selectivity of Fusion Protein Recovery From Its Molecular Variants”, Biotechnology and Bioengineering, Volume 110, Issue 7, pages 1973-1983
References: Chichi Huang, 2009, “Receptor-Fc fusion therapeutics, traps, and MIMETIBODYTM technology”, Current Opinion in Biotechnology, 20:692-699.
Hutchinson et al., 2006, “ Shear stress analysis of mammalian cell suspension for prediction of industrial centrifugation and its verification”, Biotech. And Bioeng., 95:483-491.
Kelley et al., 2009, “Downstream processing of monoclonal antibodies: Current practices and further opportunities”, In: Gottschalk U, editor. Process scale purification of antibodies. P1-23.
Prashad et al., 2006, “Depth filtration: Cell clarification of bioreactor offloads”, Filtration & Separation, 43(7):28-30.
Yigzaw et al., 2006, “Exploitation of the adsorption properties of depth filters for host cell protein removal during monoclonal antibody purification”, Biotechnol. Prog., 22:288-296.
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