20180904 poster impurity precipitation biotech day · title: microsoft powerpoint - 20180904 poster...
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0102030405060708090100
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Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6 Exp 7 Exp 8 Exp 9 Exp 10 Exp 11 Exp 12
Mono
mer [%
]
Total
HCP L
RV
precipitation + WorkBeads C10ST C10EP monomer C10 EP
502HCP [ppm] 410420503521 5001442 13032552 141228162822
Impurity Precipitation for mAb Capture OptimizationSilke Bastian, Coralie Tanzer, Anja Trapp, Jadranka KoehnRentschler Biopharma SE, Erwin-Rentschler-Straße 21, Laupheim, Germany, [email protected]
Introduction
Conclusion
A straightforward pre-purification step in the cell supernatantreduces the burden of heavy impurity loading on the initialcolumn-based process step (C10) and can help to improvecolumn performance and lifetime. To reach this, a precipitationstep can be implemented by conditioning of the startingmaterial before mAb capture. This promotes the clearance ofprocess and product-related impurities such as low and highmolecular weight species (LMWS, HMWS), host cell proteins(HCPs), chromatin and DNA from the target protein. Thus thiswork focused on the development of an efficient pre-treatmentof cell culture supernatant through precipitation prior C10 toselectively remove impurities and thereby optimized the columnperformance during mAb capture. Therefore, design ofexperiment (DoE) screenings had been performed, coveringdifferent pH ranges, precipitation agents such as inorganic salts(CaCl2, NaCl, PO43-, (NH4)2SO4), allantoin, chitosan,ethacridine, caprylic acid, polyethylene glycol as well as the useof WorkBeads™ 40 TREN (WorkBeads).
Impurity precipitation is an effective method for thepre-purification of cell supernatant and can beimplemented by conditioning of the starting materialto remove significant amounts of process andproduct related impurities already at the verybeginning of the DSP process sequence.
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Precipitation improves mAb capture
Experiment 1 2 3 4 5 6 7 8 9 10 11 12pH 7 5 7 5 7 7 5 5 5 5 5 5
CA [mM] 0 0 0 0 5 5 15 15 15 15 15 15Allantoin [%(w/v)] 0 0 0 0 0 0 0 1 0 1 1 1
CaCl2 [mM] 0 0 0 0 105 105 0 0 0 0 0 0WorkBeads load [mgmL-1] - - 50 50 - 50 - - 50 50 75 100
Figure 4: Effect ofprecipitation setups 1 -12 (s. table) on ProAcapture. HCP removal isgiven as log reductionvalue (LRV) comparedto the starting materialon the left y-axis. Themonomer content of theelution pool (C10EP) isgiven on the right y-axis.HCP contents of theC10EP is given abovethe diagram.
Early precipitation studiesInitial pretests with setups of different precipitation agents and conditions gavepromising results regarding recovery and purity of the target mAb (Fig. 1).
Precipitationconditions
pH 5 pH 7- 5 mM CA 7.5 mM CA - 5 mM CA 7.5 mM CA- 10 mM
CaCl250 mMCaCl2
10 mM CaCl2
50 mM CaCl2 - 105 mM
CaCl2145 mM CaCl2
105 mM CaCl2
145 mM CaCl2
mAb recovery[%] 96 96 94 96 94 99 92 90 91 90
HCP removal [LRV] 0.0 0.3 0.3 0.5 0.3 0.0 0.2 0.2 0.2 0.2
HMWS [%] 4.8 8.1 6.6 9.0 7.4 4.7 2.0 1.9 1.8 1.5Monomer [%] 89.9 86.2 87.9 85.7 87.3 90.1 92.3 92.0 92.4 92.5HMWS after
ProA [%] 2.0 2.5 1.7 2.4 2.1 2.0 0.9 0.9 0.9 0.9Monomer after
ProA [%] 98.0 97.5 98.3 97.6 97.9 98.0 99.1 99.1 99.1 99.1Figure 1: Precipitations of a cell culture supernatant with CaCl2, PO43- and caprylic acid (CA) were analyzed by SE-HPLC (above). Protein A purification and subsequent SE-HPLC (below) served to compare the monomer purity andHMWS content after ProA capture. With setups using 7 mM PO43- and higher concentrations of CaCl2 at pH 7,highest mAb purities and HMWS removal were reached (highlighted in light blue), while no LMWS were detectable.
Intensified screening
[1] Chen et al. 2016 Appl. Microbiol. Biotechnol. 100 (23) : 9933–9941.[2] Gagnon et al. 2014 J. Chromatogr. A 1340 : 68–78.[3] Nian et al. 2016 J. Chromatogr. A 1431 : 1–7.
Setups that had given the bestresults in all previous screeningswere repeated and the pre-purified samples were used asstarting material (C10ST) forProteinA chromatography (ProA)for mAb capture. Treatments with15 mM caprylic acid (CA) and 1 %(w/v) allantoin at pH 5 promotedHCP removal the most, wherebythe highest HCP log reductionvalue (LRV) were reached withsetups containing WorkBeads(see Fig. 4). However, benefits ofthe latter vanished after ProA.Also the monomer purity of theelution pool (C10EP) wascomparable for all setups afterthe capture step.
Cell supernatantprecipitation,
filtration
Further experiments focused on the implementation of WorkBeads during impurity precipitation (Fig. 2). WorkBeads comprise a multimodal ligand that is cationically charged belowpH 9 and have been shown to promote the removal of chromatin, DNA, HCPs, and viruses and thus reduce column fouling.[1]-[3]
Figure 2: SE-HPLC was performed to analyze the impact ofthe application of WorkBeads on precipitations. Testedsettings were 1 % (w/v) allantoin, 24 mM caprylic acid, pH 5,38 mg mL-1 load of WorkBeads. Incubation time forprecipitation was 2 h. Arrows highlight setups with decreasedHMWS (left) and LMWS (right) content in differently treatedcell culture supernatant.
Harvest Harvest + WorkBeads Precipitation + WorkBeadsPrecipitation
DoE had beenutilized to investigatesetups coveringpH 5 – 6, 0 - 15 mMcaprylic acid (CA),0 - 2 % (w/v) allantoinand 50 - 100 mg mL-1load of WorkBeadsfor pre-treatment ofthe cell culturesupernatant and hadbeen compared tothe same setupswithout the additionof WorkBeads. InFig. 3 3D contourplots of the DoE aredepicted. Figure 3: 3D contour plots of the DoE visualize recovery (left) and monomer purity (middle) as well as HCP LRV (right). Setups without
(above) and with (below) the application of WorkBeads have been investigated and are comparatively juxtaposed. As allantoin had nosignificant influence on HCP removal (right), only contour plots for setups with 0 % and 2 % (w/v) allantoin are shown representatively.Model parameters for the model fit (R²) and prediction precision (Q²) are given in grey.
R2 = 0.64, Q2 = 0.51
For the criteria of > 90 % recovery, > 83 % mAb purity and < 150 000 ppm HCP (LRV 0.5), the DoE allowed to detect auspicious trends for precipitation (pH 5, 15 mM CA) and fortreatments using WorkBeads (pH 5.3, 12 mM CA, 0.4 % (w/v) allantoin, 63 mg mL-1 load).Scale-up experiments (10 mL and 100 mL) were additionally performed for setups with the most promising outcomes to evaluate their scalability. Total recovery, monomer content aswell as HCP reduction were thereby comparable over the scales (data not shown).
mAb captureProtein A (C10)
HCP LRV
R2 = 0.94, Q2 = 0.59R2 = 0.83, Q2 = 0.68
0.10.20.30.4
0.5
Allantoin [%] = 2
5 5.2 5.4 5.6 5.8 6pH [-]
HCP LRV [-]
0.1
0.2
0.3
0.4
0.5
0.6
0.30.4
0.50.6
0.20.3
0.40.5
0.40.5
0.7
Allantoin [%] = 2
5 5.2 5.4 5.6 5.8 6pH [-]
HCP LRV [-]
0.1
0.2
0.3
0.4
0.5
0.6
0.7
HCP LRV [-]
0.30.4
0.50.6
0.20.3
0.40.5
0.40.5
0.7
Allantoin [%] = 0
048
12
048
12
048
12
5 5.2 5.4 5.6 5.8pH [-]
5R2 > 0.97, Q2 > 0.89
R2 > 0.97, Q2 > 0.91
8282.58383.584
82
82.583
83.5
84
82.583
83.5
84
Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2
02468
101214
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8 6pH [-]
Monomer [%]
81.58282.58383.58484.5
10098
99 100
96 9798 99
Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2
02468
101214
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8 6pH [-]
Recovery [%]
95
96
97
98
99
100
84.58585.58686.5
8787.5
84.58585.58686.5
8787.5
84.58585.58686.5
8787.5
8585.58686.5
8787.5
8585.58686.5
8787.5
8585.58686.5
8787.5
8686.58787.5
8888.5
8686.58787.5
8888.5
8686.58787.5
8888.5
Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2
048
12
048
12
048
12
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8 6pH [-]
Monomer [%]
84.58585.58686.58787.58888.589
95 9693
9495 92
93
94
94 9592
93
94
91 9293
93 94 9192
93
9091 92
Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2
048
12
048
12
048
12
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8pH [-]
5 5.2 5.4 5.6 5.8 6pH [-]
Recovery [%]
89
90
91
92
93
94
95
96
R2 = 0.91, Q2 = 0.83
Recovery Monomer purity