0 10 20 30 40 50 60 70 80 90 100 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6 Exp 7 Exp 8 Exp 9 Exp 10 Exp 11 Exp 12 Monomer [%] Total HCP LRV precipitation + WorkBeads C10ST C10EP monomer C10 EP 502 HCP [ppm] 410 420 503 521 500 1442 1303 2552 1412 2816 2822 Impurity Precipitation for mAb Capture Optimization Silke Bastian, Coralie Tanzer, Anja Trapp, Jadranka Koehn Rentschler Biopharma SE, Erwin-Rentschler-Straße 21, Laupheim, Germany, [email protected] Introduction Conclusion A straightforward pre-purification step in the cell supernatant reduces the burden of heavy impurity loading on the initial column-based process step (C10) and can help to improve column performance and lifetime. To reach this, a precipitation step can be implemented by conditioning of the starting material before mAb capture. This promotes the clearance of process and product-related impurities such as low and high molecular weight species (LMWS, HMWS), host cell proteins (HCPs), chromatin and DNA from the target protein. Thus this work focused on the development of an efficient pre-treatment of cell culture supernatant through precipitation prior C10 to selectively remove impurities and thereby optimized the column performance during mAb capture. Therefore, design of experiment (DoE) screenings had been performed, covering different pH ranges, precipitation agents such as inorganic salts (CaCl 2 , NaCl, PO 4 3- , (NH 4 ) 2 SO 4 ), allantoin, chitosan, ethacridine, caprylic acid, polyethylene glycol as well as the use of WorkBeads™ 40 TREN (WorkBeads). Impurity precipitation is an effective method for the pre-purification of cell supernatant and can be implemented by conditioning of the starting material to remove significant amounts of process and product related impurities already at the very beginning of the DSP process sequence. www.rentschler-biopharma.com Precipitation improves mAb capture Experiment 1 2 3 4 5 6 7 8 9 10 11 12 pH 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 15 Allantoin [%(w/v)] 0 0 0 0 0 0 0 1 0 1 1 1 CaCl 2 [mM] 0 0 0 0 105 105 0 0 0 0 0 0 WorkBeads load [mgmL -1 ] - - 50 50 - 50 - - 50 50 75 100 Figure 4: Effect of precipitation setups 1 - 12 (s. table) on ProA capture. HCP removal is given as log reduction value (LRV) compared to the starting material on the left y-axis. The monomer content of the elution pool (C10EP) is given on the right y-axis. HCP contents of the C10EP is given above the diagram. Early precipitation studies Initial pretests with setups of different precipitation agents and conditions gave promising results regarding recovery and purity of the target mAb (Fig. 1). Precipitation conditions pH 5 pH 7 - 5 mM CA 7.5 mM CA - 5 mM CA 7.5 mM CA - 10 mM CaCl 2 50 mM CaCl 2 10 mM CaCl 2 50 mM CaCl 2 - 105 mM CaCl 2 145 mM CaCl 2 105 mM CaCl 2 145 mM CaCl 2 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 1.5 Monomer [%] 89.9 86.2 87.9 85.7 87.3 90.1 92.3 92.0 92.4 92.5 HMWS after ProA [%] 2.0 2.5 1.7 2.4 2.1 2.0 0.9 0.9 0.9 0.9 Monomer after ProA [%] 98.0 97.5 98.3 97.6 97.9 98.0 99.1 99.1 99.1 99.1 Figure 1: Precipitations of a cell culture supernatant with CaCl 2 , PO 4 3- and caprylic acid (CA) were analyzed by SE- HPLC (above). Protein A purification and subsequent SE-HPLC (below) served to compare the monomer purity and HMWS content after ProA capture. With setups using 7 mM PO 4 3- and higher concentrations of CaCl 2 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 best results in all previous screenings were repeated and the pre- purified samples were used as starting material (C10ST) for ProteinA chromatography (ProA) for mAb capture. Treatments with 15 mM caprylic acid (CA) and 1 % (w/v) allantoin at pH 5 promoted HCP removal the most, whereby the highest HCP log reduction value (LRV) were reached with setups containing WorkBeads (see Fig. 4). However, benefits of the latter vanished after ProA. Also the monomer purity of the elution pool (C10EP) was comparable for all setups after the capture step. Cell supernatant precipitation, filtration Further experiments focused on the implementation of WorkBeads during impurity precipitation (Fig. 2). WorkBeads comprise a multimodal ligand that is cationically charged below pH 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 of the application of WorkBeads on precipitations. Tested settings were 1 % (w/v) allantoin, 24 mM caprylic acid, pH 5, 38 mg mL -1 load of WorkBeads. Incubation time for precipitation was 2 h. Arrows highlight setups with decreased HMWS (left) and LMWS (right) content in differently treated cell culture supernatant. Harvest Harvest + WorkBeads Precipitation + WorkBeads Precipitation DoE had been utilized to investigate setups covering pH 5 – 6, 0 - 15 mM caprylic acid (CA), 0 - 2 % (w/v) allantoin and 50 - 100 mg mL -1 load of WorkBeads for pre-treatment of the cell culture supernatant and had been compared to the same setups without the addition of WorkBeads. In Fig. 3 3D contour plots of the DoE are depicted. 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 no significant 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 for treatments 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 as well as HCP reduction were thereby comparable over the scales (data not shown). mAb capture Protein A (C10) HCP LRV R2 = 0.94, Q2 = 0.59 R2= 0.83, Q2 = 0.68 0.1 0.2 0.3 0.4 0.5 Allantoin [%] = 2 5 5.2 5.4 5.6 5.8 6 pH [-] HCP LRV 0.1 0.2 0.3 0.4 0.5 0.6 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.4 0.5 0.7 Allantoin [%] = 2 5 5.2 5.4 5.6 5.8 6 pH [-] HCP LR 0.1 0.2 0.3 0.4 0.5 0.6 0.7 HCP LRV [-] 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.4 0.5 0.7 Allantoin [%] = 0 0 4 8 12 0 4 8 12 0 4 8 12 5 5.2 5.4 5.6 5.8 pH [-] 5 R2 > 0.97, Q2 > 0.89 R2 > 0.97, Q2 > 0.91 82 82.5 83 83.5 84 82 82.5 83 83.5 84 82.5 83 83.5 84 Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2 0 2 4 6 8 10 12 14 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 6 pH [-] Monome 81.5 82 82.5 83 83.5 84 84.5 100 98 99 100 96 97 98 99 Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2 0 2 4 6 8 10 12 14 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 6 pH [-] Recover 95 96 97 98 99 100 84.5 85 85.5 86 86.5 87 87.5 84.5 85 85.5 86 86.5 87 87.5 84.5 85 85.5 86 86.5 87 87.5 85 85.5 86 86.5 87 87.5 85 85.5 86 86.5 87 87.5 85 85.5 86 86.5 87 87.5 86 86.5 87 87.5 88 88.5 86 86.5 87 87.5 88 88.5 86 86.5 87 87.5 88 88.5 Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2 0 4 8 12 0 4 8 12 0 4 8 12 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 6 pH [-] Monom 84.5 85 85.5 86 86.5 87 87.5 88 88.5 89 95 96 93 94 95 92 93 94 94 95 92 93 94 91 92 93 93 94 91 92 93 90 91 92 Allantoin [%] = 0 Allantoin [%] = 1 Allantoin [%] = 2 0 4 8 12 0 4 8 12 0 4 8 12 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 pH [-] 5 5.2 5.4 5.6 5.8 6 pH [-] Recove 89 90 91 92 93 94 95 96 R2 = 0.91, Q2 = 0.83 Recovery Monomer purity