High Throughput Optimization Approach for Single Step Polishing
of Monoclonal Antibodies Post Protein A Capture
Aleksandar Cvetkovic1, Amitava Kundu2 and René Gantier1
1Pall Life Sciences, Westborough, MA, USA; 2Genmab MN, Inc., Brooklyn Park, MN, USA
inTRoduCTion
Mixed‐mode chromatography is a flexible and cost‐effective technique applicable to intermediate and polishing steps in monoclonal antibody (MAb) purification. We used various mixed‐mode sorbents to develop a single polishing step of MAbs following Protein A
capture. High‐throughput experimentation (HTE) tools were employed to select conditions in both bind/elute and flow through modes to be transferred to column chromatography. Mixed-mode sorbents used in flow‐though (FT) mode (HEA HyperCel™ sorbent, Pall
Life Sciences) provided good contaminant (aggregates and HCPs) clearance with MAb loads up to 150 mg/mL of sorbent. This study confirms the applicability of mixed‐mode chromatography for post‐Protein A purification of MAbs.
mixEd‐modE ChRomaTogRaphy:
mEp, hEa and ppa hypERCEl soRbEnTs
sElECTion oF FT modE opERaTion CondiTions using hTE
O
CH2 CH2 CH2 S
CH2 CH2
H+
N CH2 (CH2)4 CH3
(Spacer)
HEA HyperCel Sorbent
Does not meet requirements
MEP HyperCel Sorbent
20
15
PPA HyperCel Sorbent
30
30
25
25
20
15
20
15
10
10
10
5
5
5
3.5
40
4.5
5.0
5.5
6.0
3.0
pH
3.5
40
4.5
5.0
5.5
6.0
3.0
3.5
40
pH
4.5
5.0
5.5
6.0
pH
Operation Space Criteria: (red) MAb yield > 80%; (green) FT HCP Content < 25 ppm; (blue) FT Aggregate Content <1%
CH2 CH2
hEuRisTiCally dEduCEd mab load impaCT on
pERFoRmanCE oF mixEd-modE soRbEnTs
Figure 5
Schematic Representation of the Heuristic Approach
Implemented on the Experimental HTE Data Collected
on HEA Hypercel Sorbent at 5 mg MAb per mL Load
and the Results Obtained Using Same Approach for
MEP and PPA Hypercel Sorbents
Figure 2
Mixed-mode Chromatography Optimization Strategy
HEA HyperCel
30
ad
pH
Lo
Buffer
Sorbent bed
Filter plate
2. Screening on 96-Well Plates
• AcroPrep™ Advance Filter Plates
• AcroPrep ScreenExpert Plates
• Vacuum manifold
sorbents: MEP, HEA, PPA HyperCel
Table 2
HTE Evaluation Conditions
20
15
5
3.0
3.5
40
4.5
pH
5.0
5.5
6.0
FT yield >60%, HCP <10 ppm, Agg. <0.75%
30
AU
0.004
0.002
20
15
Optimal conditions
Wash: pH 4.4,
29.2 mS/cm
10
Min
11
12
13
14
Elution pH
300-350
250-300
7.5
200-250
7.0
6.5
150-200
6.0
5.0
4.0
4.2
4.4
4.6
4.8
5.0
Load pH
5. Transfer to Column
MAb FT Yield (%)
30
10
MAb Yield %
80
100
FT HCP, ppm
0
25
Aggregates %
0
1
25
Conductivity
Conductivity
15
3.5
40
4.5
pH
5.0
5.5
5.5
6.0
PPA HyperCel
20
15
10
5
3.0
6.0
20
Experimental conditions
Pall 1 mL PRC prepacked columns (0.5 cm I.D.,
5 cm bed height)
4 minutes residence time
Load sample: up to 150 mg MAb per mL of sorbent
3.5
40
4.5
pH
5.0
5.5
6.0
Results
HEA HyperCel sorbent - best aggregate removal
performance (Figure 6A)
HEA HyperCel sorbent – also good HCP clearance
and MAb yield even at 150 mg MAb load per mL
of sorbent presented (Table 4 and Figure 6B)
Figure 6
Transfer of Optimized HTE Conditions on Column
A
HEA,
PPA and
B
MEP HyperCel
Aggregate Content (C/C0), %
9
8
Aggregate Content (C/C0), %
0
5.5
20
5.0
0.001
4. Result Analysis
• Design space for optimum performances
25
4.5
pH
TRansFER oF opTimizEd FT modE CondiTions To Column ChRomaTogRaphy
0.003
8.0
<1
1 – 10
10 – 20
20 – 30
30 – 40
40 – 50
50 – 60
60 – 70
70 – 80
80 – 90
90 – 99
> 99
40
0.005
HCP (ppm)
MAb Yield (%)
30
3.5
0.006
8.5
30
3.0
25
10
3. Analytical Testing
• Aggregate quantification (SEC HPLC)
• HCP quantification (ELISA)
• MAb elution yield (Poros A, OD280nm)
Determination conditions for FT
steps was based on an axial point
response surface DoE (Minitab◆)
15
5
Predicted responses
mAb Yield > 90 %
HCP < 10 ppm
Aggregates < 0.5%
25
3.0
MEP/HEA/PPA HyperCel
5 mg/mL of sorbent
Low pH
High pH
3 to 6
6 to 9
4 to 30 mS/cm
9
3
17 mS/cm
20
10
5
Conditions Tested
25
100
80
60
40
20
0
0
50
100
150
MAb Load (mg/mL)
HEA HyperCel
30
100
25
80
20
60
15
40
10
20
5
0
0
150
0
50
100
HCP Content (ppm)
235 ppm (ng/mg MAb) host cell
proteins (HCP)
Table 3
Optimal Conditions for FT Processing of MAb Determined
by HTE Study
Sorbent
pH
Conductivity, mS/cm
MEP HyperCel
4.5
5.1
PPA HyperCel
3.9
4
HEA HyperCel
4.4
29.3
Conductivity
1.9% aggregate
Optimal conditions
Wash: pH 4.5,
12.4 mS/cm
10
Elution Cond
1. Design of Experiment (DoE)
• Critical parameters (loading and elution conditions)
• Quality attributes (yield, HCP and aggregates)
30
25
Elution pH
MAb concentration: 19.5 mg/mL
MEP HyperCel
FT yield >80%, HCP <25 ppm, Agg. <1%
Conductivity
opTimizaTion oF mixEd-modE soRbEnTs FoR posT pRoTEin a
sTEp mab puRiFiCaTion
Conductivity
(Spacer)
PPA HyperCel Sorbent
Conductivity
H+
N CH2
pKa ~ 8
Load Conductivity
Elution pH
Elution Conductivity
HEA HyperCel Sorbent
3.0
pKa ~ 8
Alternative/complement to conventional
HIC or hydroxyapatite
Sorbent
Protein Load
Load pH
25
PPA HyperCel sorbent:
Alternative to ion exchange when
feedstock is directly loaded (no
dilution or UF/DF)
(pI 7.6)
HEA - better flexibility
pKa = 4.8
Alternative to traditional methods
(ion exchange, HIC, affinity…)
load: Post Protein A elute MAb
Selectivity for MAb vs. aggregates
30
Conductivity
MEP HyperCel Sorbent
Broad affinity for HCPs
Conductivity
Meet operational requirements
Figure 1
Ligand Structure of HyperCel Mixed-Mode Sorbents
Mixed-mode chromatography exploits
multiple, distinct protein-ligand interactions
to adsorb target proteins or impurities.
Mixed-mode chromatography offers new
solutions to separations:
Figure 4
Identification of Sweet Spot pH and Conductivity in FT Under Low pH Load
Conditions
Conductivity
MEP and HEA HyperCel sorbents:
MAb Load (mg/mL)
Table 4
Best Column Performance: HEA HyperCel Sorbent (150 mg/mL MAb Load)
15
5
3.0
3.5
4.0
4.5
pH
5.0
5.5
10
HCP (ppm)
30
FT HCP (ppm)
<1
1–5
5 – 10
10 – 15
15 – 20
20 – 25
25 – 50
50 – 100
> 100
25
Conductivity
Sweet spot cond.
Yield >80%
HCP <25 ppm
Aggregates <1%
6.0
20
15
5
3.0
3.5
4.0
4.5
pH
5.0
Optimizer Minitab
10
Optimal
High
D
0.62446 Cur
Low
5
3.0
3.5
4.0
4.5
pH
5.0
5.5
6.0
30
< 0.1
0.1 – 0.5
0.5 – 1.0
1.0 – 2.0
2.0 – 4.0
4.0 – 6.0
6.0 – 8.0
8.0 – 10.0
> 10.0
20
15
FT, %
Targ: 99.0
y = 90.1255
d = 0.53292
HCP, ppm
Targ: 1.0
y = 8.3630
d = 0.69321
10
5
3.0
3.5
4.0
4.5
pH
5.0
5.5
6.0
Aggregate %
Targ: 0.10
y = 0.4068
d = 0.65917
Contact: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • +65 6389 6500 (Asia/Pacific) • E-mail: biopharm@pall.com • Web: www.pall.com/biopharm
Aggregate
< 0.5%
HCP
23 ppm
ConClusions
Optimal load conditions:
Wash pH 4.3
Cond. 12.4 mS/cm
Aggregates %
25
Conductivity
Cond
30.0
[12.3626]
4.0
MAb Yield
94.9%
6.0
Composite
Desirability
0.62446
Aggregates (%)
Figure 3
Data Processing Approach Used
to Determine Optimal Operation
Conditions (MAb Yield of Recovery
and Purity) Using HEA Hypercel
Sorbent in FT Mode
pH
6.0
[4.5057]
3.0
5.5
Conductivity
29.3 mS/cm
pH
4.3
Predicted responses:
MAb Yield = 91.3 %
HCP = 5.5 ppm
Aggregates = 0.4%
High Throughput Screening on AcroPrep™ 96-well sorbent plates allows fast process optimization
Use of Pall mixed-mode sorbents in FT mode after Protein A step provides excellent separation performance
– High yield of recovery
– Efficient contaminant removal (aggregate, HCP)
– High MAb load g reduced sorbent volume
– HEA HyperCel sorbent provided best performance with specific MAb
• ~ 95% MAb yield at 150 mg/mL MAb load
• < 0.5% aggregate and < 25 ppm HCP
Mixed-mode sorbents operated in FT mode are a good alternative to high capacity IEX sorbents
© 2014, Pall Corporation. Pall,
, Acrodisc, AcroPrep, and HyperCel are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. ◆Minitab is a trademark of Minitab, Inc. 7/14, GN14.9444