ROF SEIGETARTS EVITAVONNI :ERUPXBAM PESIAD NOITACIFIRALC NI ECNARAELC PCH GNICNAHNE
AND DOWNSTREAM PROCESSING OF MABS
INTRODUCTION
In recent years, biopharmaceutical manufacturing has shown
titers generally associated with higher cell densities. These increases in productivity induces higher levels of process-related impurities like host cell proteins (HCP), DNA, HMW fragments or lipids and consequently shifts the production bottleneck to the downstream
In such context,
limit and will not be able to provide enough protection to the .
TECHNOLOGY
DAISEP MabXpure™
(SUT) combines multimodal depletion mechanisms (Size Exclusion and Anion Exchange) to improve bioburden depletion performances. MabXpure can be used as a single device technology or within a
the resin with very low ratio feed/media, or as a polishing cartridge, when prepacked, without compromising the antibody yield (>90%).
DNA and 1 LRV for HCP under static or dynamic conditions.
as a prepacked cartridge, it is used as a Protein A guard column or as ion exchange DSP step combination. MabXpure improves overall
formulation.
CONCLUSION
HCPs and DNA depletion technology. The use of MabXpure as a
of use are optimal with high mix ratios and short contact times for sample preparation, or lower mix ratios and higher contact times for
MabXpure has a very high capability for depleting host cell proteins and DNA, with extremely high mAb recovery (>95%). Less intrusive
to the product, it allows the removal of up to 1 LRV of HCPs, 2 LRV of DNA depending on the mode of action, and reduces co-eluted HCPs species. MabXpure can supplement, complement, or replace the pre-existing DSP unit operations.
Contact us at [email protected]
MECHANISM OF INTERACTION &
DEPLETION
Small moleculesnucleic acids
HCDNA
HCP
SEC
AEX mAb
SEC
mAb
Small moleculesnucleic acids
HCDNA
HCPs
AEX
DAISEP MabXpure
HCP 27kDa pI 5.8
AEX
DNA 100-300kDa pI 5.5
AEX-SEC
IgG 150kDa pI 8.3
SEC
• SEC prevents antibodies from accessing the interiorof the MabXpure bead. The antibodies remain in the
• HCPs, DNA fragments, and small molecules arecaptured by AEX.
• The highly porous structure brings high-bindingcapabilities with multimodal adsorption features.
SEC (Size Exclusion) and AEX ( Anion Exchange)
$0
$100,000
$200,000
$300,000
$400,000
$500,000
$600,000
$700,000
$800,000
$900,000
$1,000,000
initial capex/10 Total cost/batch Total cost/year/10 Cost/kg mAb
MabXpure as Filter Aid
99 70 79 63
0
50
100
150
Time/batch (hrs)
$3M
$.2M
$30%
$1M
2+ day
COGS
• Various scenarios of MabXpure implementation are possible but the economical savings evaluation ishighly linked to the process steps in place.
• Economic improvements are possible for capital expenditure and total cost per batch and gram of mAb.• The use of MabXpure FT can speed-up the production time and save labor.
–$30%
Feed 1: HCP: 800 mg/mLHMW: 6%
Feed 2: HCP: 200 mg/mLHMW: 8.8%
Feed 1: HCP: 17 mg/mLHMW: 0.6%
Feed 2: HCP: <1.6 mg/mLHMW: 1%
Post ProA step HCP: <50 ppm HMW: 0.5%
Feed 1: HCP: 800 mg/mLHMW: 6%
Feed 2: HCP: 200 mg/mLHMW: 8.8%
Feed 1: HCP: 17 mg/mLHMW: 0.6%
Feed 2:
HCP: <1.6 mg/mLHMW: 1%
Post ProA stepHCP: <50 ppm HMW: 0.5%
Feed 1: HCP: 800 mg/mLHMW: 6%
Feed 2: HCP: 200 mg/mLHMW: 8.8%
Feed 1: HCP: 17 mg/mLHMW: 0.6%
Feed 2 : HCP: <1.6 mg/mLHMW: 1%
Post ProA stepHCP: <50 ppm HMW: 0.5%
OPTIMIZED CLARIFICATION PLATFORM
• rth,
• Orthogonal depletion mechanisms are simultaneously taking place during the alluvial
• After proteinA, the platform is reaching the drug substance criteria for HCP, DNA, HMW…•
100,000
1,000,000
10,000,000
100,000,000
total HCP HCP1 HCP2 HCP3 HCP4 HCP5 HCP6 HCP7 HCP8
Uni
t of
abu
ndan
ce
Post ProteinA
Post Mixed-Mode
Post ProteinA post MabXpure
Post Mixed-mode post MabXpure
Limit of identification
CO-ELUTED HCP DEPLETION PERFORMANCES
• MabXpure FT removes co-eluted HCPs that are not removed by classic technologies.•• MabXpure FT induces purer and safer formulations for the mAbs originally containing co-eluted HCPs.
43
•
• MabXpure depletes HCP and DNA, under staticconditions, by mixing the mAb-containing feedwith the resin.
• ed to recover a
•for impurity capture and polishing purposes.
• MabXpure FT depletes HCP and DNA under dynamicconditions from a mAb-containing feed.
• Traces of co-eluted HCPs and remaining DNA can ber
implementation
UPSTREAM DOWNSTREAM HARVEST FILL & FINISH
BIOREACTOR
CENTRIFUGE
ADF/CF
DFF PA CEX AEX
TFF
UPSTREAM HARVEST DOWNSTREAM FILL & FINISH
Objectives:•• n platform.• Target the capture of co-eluted HCPs all along the process that jeopardize the stability of
the formulation.
PROCESS SCHEME OF MABXPURE IMPLEMENTATION
43
2
1
5
MABXPURE AS FILTER CARTRIDGE (DYNAMIC PERFORMANCE)
• Dynamic conditions expose allspecies to MabXpure’s por ousstructure improving the depletion ofimpurities.
• >75% HCP depletion can beachieved with 90–100% mAbrecovery.
• MabXpure is designed to work with
improve HCP removal.
12%
20% 22%
36%
23%
100%
11% 18%
25%
52%
26%
10%
21%
39%
75%
35%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 1-5cvs 5-10cvs 10-15cvs 15-20cvs Pool
1' 2' 5'
90% 100% 100% 100% 98% 96%
76%
93%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 1-5cvs 5-10cvs 10-15cvs 15-20cvs Pool
1' 2' 5'
HCP Depletion Performances vs residence time mAb Recovery Performances vs residence time
CHO-K1, DNA 1.000 ng/mL, HCP 120 µg/mL, IgG 2 mg/mL
•
Phosphate ions do not impact the depletion.• Thanks to the AEX mechanism, virus clearance can
be claimed and up to 5 LRV for MVM and MuLV canbe reached.
• HCP depletion is observed after classic chromatographic steps of a
• With 3 chromo steps, MabXpure FT helps to streamline the process
• For a two-step process MabXpure can allow the removal of a bindand elute step.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed No DF pH 7.2
50mM Na Acetate pH 5.6
50mM NH4OAc
pH 6.5
50mM Tris HCl pH 7.5
HCP Depletion Performances vs Diafiltration Buffer
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
PostProA
PostMM
PostAEX FT
HC
P L
RV
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
PostProA
PostMM
HC
P L
RV
HCP Depletion Performances vs 2 & 3 Steps DSP platform
0
1
2
3
4
5
6
1:10 1:25 1:50
LRV
Viru
s
MVM MuLV
Virus Clearance with MabXpure FT
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed No DF pH 7.2
50mM Na Acetate pH 5.6
50mM NH4OAc
pH 6.5
50mM Tris HCl pH 7.5
HCP Depletion Performances vs Diafiltration Buffer
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Post ProA
Post MM
Post AEX FT
HC
P L
RV
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Post ProA
Post MM
HC
P L
RV
HCP Depletion Performances vs 2 & 3 Steps DSP platform
0
1
2
3
4
5
6
1:10 1:25 1:50
LRV
Viru
s
MVM MuLV
Virus Clearance with MabXpure FT
CHO-K1, DNA 1.200 ng/mL, HCP 460–680 µg/mL, IgG 2.7–2.8 mg/mL
44
3
3
2
5
7 7 MabXpure as Filter Aid (Staticperformance)
100%
9%
60%
7% 9% 12%
37% 35%
69%
2% 5%
9%
23% 26%
88% 83%
97% 100% 98%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 1:5 Silica
1:5 DE
1:5 1:10 1:20 1:50
HCPs DNA IgG
Depletion Performances vs mix ratio (resin:sample)
37%
27% 24%
100%
23%
3% 1%
98% 99% 99%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 1:50 5'
1:50 30'
1:50120'
HCPs DNA IgG
Depletion Performances vs contact times
100%
45% 42% 44% 41%
97% 96% 97% 94%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 6,5 7 7,4 8
HCPs IgG
100%
39% 35%
41%
93% 96% 98%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 0,5 2 4
HCPs IgG
100%
16% 17% 24%
20%
99% 97%
77%
95%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed IgG1 IgG2 IgG3 IgG4
HCP IgG
100%
15% 15% 15% 23%
95%
69% 68%
78%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed Goat pH5 Rabbit pH5
Mouse pH5
Bovine pH6.7
HCP IgG
Depletion Performances vs Feed pH Depletion Performances vs IgG subclass
Depletion Performances vs mAb titer (g/L) Depletion Performances vs IgG origin
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed IgA 5 IgM 7.4 IgM 5
HCP IgG DNA
Depletion Performances vs IgG types
100%
45% 42% 44% 41%
97% 96% 97% 94%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 6,5 7 7,4 8
HCPs IgG
100%
39% 35%
41%
93% 96% 98%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed 0,5 2 4
HCPs IgG
100%
16% 17% 24%
20%
99% 97%
77%
95%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed IgG1 IgG2 IgG3 IgG4
HCP IgG
100%
15% 15% 15% 23%
95%
69% 68%
78%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed Goat pH5 Rabbit pH5
Mouse pH5
Bovine pH6.7
HCP IgG
Depletion Performances vs Feed pH Depletion Performances vs IgG subclass
Depletion Performances vs mAb titer (g/L) Depletion Performances vs IgG origin
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Feed IgA 5 IgM 7.4 IgM 5
HCP IgG DNA
Depletion Performances vs IgG types
• Various process conditions (e.g., titer,pH) can be used without impactingbioburden depletion and mAb recovery.
• MabXpure insures a robust HCPs/DNAdepletion platform.
• All antibodies subclasses, types andorigins can be used with MabXpure.
• Adaptation of pH is required for low IgG’s isoelectric point and balance between HCP depletion and IgG recovery must be assessed.
•bioburden depletion (HCPs and DNA) and mAbrecovery.
• MabXpure guarantees combined performance for
• Flexibility of MabXpure resides in the mix ratio to beused and the time of contact.
• Increasing contact time improves bioburdendepletion.
• MabXpure adapts to your needs with contact timeand mix ratio.
• >1 LRV for HCPs and 2 LRV for DNA can beobtained.
MABXPURE AS FILTER AID (STATIC PERFORMANCE)
CHO-K1, DNA 1.000 ng/mL, HCP 180 µg/mL, IgG 2 mg/mL
SANOFI CASE STUDY
SANOFI CASE STUDY
