Performance of the AbSolute High Cap resin in the Nimotuzumab capture step

Transcription

1 Research Article Journal of Biotechnology and Bioengineering Volume 1 Issue 1 Open Access Performance of the AbSolute High Cap resin in the Nimotuzumab capture step Martinez Rosario 1*, Ruland Yvan 2, Zhao Gang 3, and Shen Chunjuan 4 1 The Center for Molecular Immunology, Cuba. 2, 3, 4, NOVASEP Asia- Biopharma Business Unit, Shanghai, China. * Corresponding author: Martinez Rosario, The Center for Molecular Immunology, Cuba. rosario@cim.sld.cu Citation: Martinez Rosario (2017) Performance of the AbSolute High Cap resin in the Nimotuzumab capture step: Nessa Journal Biotechnology and Bioengineering. Received: February 10 th 2017, Accepted: March 7 th 2017, Published: May 22 th 2017 Copyright: 2017 Martinez Rosario, Yvan Ruland, Zhao Gang and Shen Chunjuan et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Protein A is the affinity chromatography ligand of choice for first-step capture in the purification of mabs as its high selectivity gives excellent purity with high yields. Furthermore,a Protein A capture acts as a key volume reduction step in the process since it concentrates significantly the product stream. The relatively high cost of suchaffinity resins leads to consider different operational strategiesin order to well optimize this step. One of them is to evaluate Protein A matrixes with highperformance in terms of dynamic binding capacity (DBC) at high flow rates. Recently a new Protein A ligand, AbSolute High Cap (AbSolute HC), was introduced by Novasep Company, which is modified with respect to high binding capacity at higher velocities. This article describes the performance of AbSolute HC gelfor Nimotuzumab capture, by measuring its Dynamic Binding Capacity (DBC) at different flow rates and feed concentrations at lab scale. AbSolute HC data are also compared with MabSelectSuRe, the affinity gel used in the current purification process. The process conditionsare adjusted including the maximum speed for all steps with an assessment of impact on Nimotuzumab purity using AbSolute HC. In addition the impact of loading amount on aggregates formation with this media and the resin lifetime are evaluated. The results show a better DBC at 10% on AbSolute HC than MabSelect Sure, with a higher DBC at 10% on Absolute HC with IgG concentration at 2.3 g/l than 0.13 g/l. No significant loss of purity or yield for speeds until 1000 cm/h for all steps is observedusing AbSolute HC resin. The lifetime of the media is tested up to 200 cycles with suitable results using NaOH and PAB solutions as CIP buffers. Key words: Protein A, mabs, affinity chromatography, AbSolute HC, dynamicbinding capacity (DBC), lineal flow rate, yield, aggregates. Nessa Publishers Page 1

2 Introduction The capture step in the downstream processing of monoclonal antibodies (mabs) is often the bottleneck and the most expensive step due to the use of Protein A media. Selection of a most suitable affinity resin based on binding capacity and affinity is typically performed prior to optimization. In recent years, several new-generation Protein A media have been launched on the market claiming improved mab capture, or improved resistance to cleaning agents. AbSolute High Cap, the new Protein A media by Novasep, developed for the capture of monoclonal and polyclonal antibodies from large volume fermentation feedstock, maintains excellent DBCs at high flow rates, therefore providing substantially improved productivity and strongly reduced costs compared with all existing protein A media. AbSolute HC is based on a modified silica matrix that exhibits strong mechanical, thermal and chemical stability. The particles are small (35 μm) with a perfect spherical shape and very narrow size distribution. Pore size is 100 nm and shows narrow size distribution (± 15 nm). Optimized particles and pores allow high efficiency and faster mass transfer which increase the Dynamic Binding Capacity (DBC) (1). Particles Cross Section Surface of Particle Figure 1: Pictures of AbSolute High Cap beads The matrix is rigid and incompressible so a 1L column is packed with 1L of AbSolute HC and the media does not shrink or swell in different solutions. These exceptional mechanical properties combined with a unique kinetic performance allow operations at higher velocities (up to 1,500 cm/h, depending on the column size and equipment). Its resistance to high ph levels makes it stable through alkaline cleaning and sanitization. Indeed, AbSolute HC remains stable after 400 cycles of use with alkali washing using 100 mm NaOH M NaCl every 10 cycles (2). Also Novasep reports that using AbSolute HC gel implies an optimization of the geometry of all columns, thus minimizing the volume of protein A required to perform the step, in order to maximize productivity. In these conditions, performing the step at industrial scale using AbSolute High Cap allows a volume reduction in the media and an increase in productivity by up to 2.7-fold compared with other industry standards, resulting in a cost savings of up to $1,000,000 for the first load of media (2,3). Nessa Publishers Page 2

3 This study outlines the performance of AbSolute HC resin for Nimotuzumab capture by measuring its DBC at different flow rates and feed concentrations at lab scale. AbSolute HC data are also compared with Mab Select SuRe, current affinity gel used in the purification process. The impact of mobile phase velocity and of loading amount on product quality is evaluated as well, with the product critical quality attributes (CQA s) being measured by yield, host cell protein (HCP) clearance, and percent of aggregate. This report also evaluates the lifetime of the media, tested up to 200 cycles using NaOH and PAB solution (Phosphoric acid, Acetic acid, Benzyl alcohol mixture). Lifetime is assessed by measuring the variation of DBC over the time, but product quality in terms of purity is monitored as well over the whole lifetime study. Materials and Methods The supernatant is obtained from a perfusion bioreactor of 1000 L of mammalian cell culture. The harvests filtrated are tested at 0.13 g/l IgG concentration, and at 2.3 g/l IgG concentration after TFF concentration. Absolute HC and Mab Select Sure resins are packed into columns of 0.5 cm of internal diameter (i.d). Column height is 10 cm for all experiments except for lifetime study which is carried out with a 5 cm height column. The equipments used for this study, performed in NovaSep facilities in Shanghai, are listed in the Table 1: Table 1: List of Equipments used Equipment Supplier AKTA Purifier Analytical HPLC Tricorn 5 mm ID columns (5 and 10 cm height) Balances SDS Page imaging system Tanon-2005 Microplate reader Spectramax 190 ph meter/conductimeter Membrane TangenX TFF skid GE Healthcare Waters GE Healthcare Sartorius Tian Neng MD Mettler Toledo Novasep Novasep Buffers used at the different steps are shown in Table 2: Nessa Publishers Page 3

4 Table 2: Buffers and step in the experimentation Step Buffer Loading Nimotuzumab harvests filtrated Equilibrium 50 mm Tris 150 mm NaCl, ph , Cond ms/cm Washing 1 50 mm Tris 1 M NaCl, EDTA 10mM, ph , Cond ms/cm Washing 2 Elution Wash Cleaning Storage 50 mm Tris, 150 mm NaCl, ph , Cond ms/cm 0.1 M Citric acid, ph 3.2~3.8, Cond 3-7 ms/cm Purified water NaOH 100 mm 20 % ethanol Experimental Conditions Analytical Assays: Table 3 summarizes the analytical assays, samples, methods and equipment used in the experimentation. Nessa Publishers Page 4

5 Table 3: Analytical assays evaluated Analytical Sample Method Equipment/column/reagent items Column: Zorbax SB 300 C8, Feed, HPLC-RP 4.6*250mm,5µm Flow through/wash HPLC: waters alliance IgG for BTC test Solvents: TFA/ACN; Gradient concentration elution Spectrophotometer Eluted IgG UV280 Supplier: APL instrument Reduced SDS- PAGE Tanon EPS 300 electrophoresis system Tanon 2500 gel Image system Purity Eluted IgG Column: TSK G3000SWxl SEC 7.8*300mm HPLC: Waters Alliance ELISA Kit: Cygnus F220. HCP Eluted IgG ELISA Equipment: Molecular Devices, Spectra Max 190 Protein A ELISA Kit: Cygnus F400 leakage Eluted IgG ELISA Equipment: Molecular Devices, All the samples were filtered by 0.2 µm on sterile bottle and stored at 4 C. Spectra Max 190 Nessa Publishers Page 5

6 Breakthrough curves (BTC s) at different flow rate Experimental conditions: The buffers and steps used for those experiments are listed in table 2 above. A characterization of the capture step of Nimotuzumab is performed at lab scale from a cell culture supernatant, by tracing breakthrough curves under different conditions of loading linear velocities. Feed is loaded continuously onto the column, collections are made at regular interval of time at the column outlet. The concentration in the breakthrough C is measured by HPLC-RP (see table 3 above) and the dynamic binding capacities Q (x-axis, mg/ml gel) are calculated using the formula: Q = C0 * t * F / CV Where: Q: Dynamic binding capacity (mg/ml resin). C0: Initial protein concentration (mg/ml). t: Time (min). F: Volumetric flow rate (ml/min). CV: Column volume (ml). When the concentration C in the breakthrough is approaching the initial concentration C0, the column is fully saturated. It is washed until the UV baseline is reached and then eluted. A regeneration and equilibration is performed before the next BTC. Beside the concentration C in the breakthrough, the concentration is also measured in the Wash stream and Elution stream. Impact of speed on purity on AbSolute HC Experimental conditions: The buffers and steps used for those experiments are listed in table 2 above. A total of 10 experiments are performed, by varying the mobile phase velocity between 300 and 1000 cm/h. In some experiments the velocity is kept the same for all steps (load, wash, elution, CIP), in others the velocity is increased for the wash or elution step. The time for loading step is adjusted so that 60 to 80 % of the total capacity previously determined in BTC s experiments is reached. After elution, the product is incubated during 1 hour at low ph condition (elution buffer at ph ) at room temperature for viral inactivation. Then a microfiltration using 0.22 µm filter is performed to reduce bioburden content. Nessa Publishers Page 6

7 Before SEC, SDS Page, HCP and Protein A analysis (see table 3), the samples collected at elution step are desalted using following conditions: Column: Hitrap 5 ml; Resin: Sephadex G 25; Buffer: 20 mm Tris, ph , conductivity ms/cm; Flow rate: cm/h; Loading volume: % column volume. Lifetime studies Experimental conditions: The lifetime study is carried out on a Tricorncolumn of dimension 5 x 50 mm (GE Healthcare) packed with 1 ml of AbSolute HC. 200 cycles are performed using the buffer conditions listed in table 2 except the cleaning in place (CIP) conditions, a mixture of acids and alcohols PAB being used instead of NaOH. PAB is a mixture of 120mM H3PO4, 167 mm acetic acid and 2.2 % benzyl alcohol, applied on the column for cleaning at each cycle with 20 minutes contact time. Every 10 cycles, NaOH CIP is performed as well using a 0.1 mm NaOH solution with 20 minutes contact time as well. When NaOH CIP occurs, the column is washed in between the PAB and the NaOH cleaning steps. All conditions are summarized in table 4 below. Table 4: Gel Lifetime conditions used Step Solution Flow ph Conductivity CV rate (ms/cm) PAB PAB: 45 cm/h ~1.5-3 [a] 120 mm H3PO4, Hygienization 167 mm acetic acid, 2.2% benzyl alcohol NaOH CIP [b] 50 mm Tris-HCl, 150 mm /Wash NaCl cm/h 7.5 NaOH CIP [a] 0.1M NaOH, 1M NaCl 60 cm/h 12-4 /CIP [a] PAB hygienization is done for all the 200 cycles. Every 10 cycles, a NaOH CIP is performed as well. Contact time is 20 minutes in both cases. [b] Column is washed in between PAB and NaOH CIP. Nessa Publishers Page 7

8 A series of test is performed to check the column performance over the time, as summarized in table 5 below: Every 20 cycles starting from cycle 1, a BTC is performed under conditions described previously in this section, in order to measure the variation of 10 % DBC value along the 200 cycles. The elution stream is analyzed for Nimotuzumab purity every 20 cycles starting from cycle 20. The product eluted at cycles 31, 33, 35, 37 and 39 is also analyzed, in order to evaluate the impact on quality of Nimotuzumab when only PAB solution is used as CIP test, and no NaOH. Table 5: tests performed during lifetime study with identification of corresponding cycle number Results and Discussion BTC test at different flow rate on MabSelect Sure and AbSolute HC. The quantity of antibodies that can be loaded per ml of gel is determined by the breakthrough curves performed at the linear velocities that maximize the overall process productivity. The breakthrough curves of the resins studied are presented in figures 2 and 3 at the linear flow rate defined and feed concentration C0 of 0.13 and 2.3 g/l IgG. Nessa Publishers Page 8

9 Figure 2: BTC of MabSelect Sure resin at 0.13 and 2.3 g/l IgG in feed. Figure 3: BTC of AbSolute HC resin at 0.13 and 2.3 g/l IgG in feed. In all cases, increasing the linear flow rate decreases the dynamic binding capacity. This is mainly due to the fact that using a faster flow rate increases the resistance to the mass transfer of the protein to the interior adsorption sites of the matrix beads. Antibodies are very large proteins of low diffusivity in the mobile phase and at higher speed, a larger number of them will not be able to enter the pores in the matrix beads. As a result of a low diffusivity, it will also take a longer time for the proteins inside the pores to get out of the bead and undergo further elution along the column. The higher is the mobile phase speed, the shallower is the adsorption front on the breakthrough curve and the higher is the concentration of antibody in the breakthrough for a given loading (4,5). Numerous chromatographic models have been well described in the literature, to characterize the adsorption equilibrium and adsorption kinetics (6,7). In the case of AbSolute adsorbent, a modeling approach based on integration of process modeling and experimentation has been applied and has shown the high capacity properties of such Protein A adsorbent based on matrix silica (8). As a matter of fact it is remarkable in the figure 3 that the breakthrough curves in AbSolute HC gel are steep whatever the linear velocity, due to a very uniform particle size distribution and a homogeneous pore size distribution for a better mass transfer. Nessa Publishers Page 9

10 On the same figure we observe on the BTC s achieved with MabSelect SuRe resin some difficulties to reach the full saturation. This has been reported for many other Protein A adsorbents (9) with some suggestions related to molecular stretching and changes in transport mechanism upon adsorption, broad particle size distribution, competition between monomers and aggregates. The results as shown on figure 3 could support the explanations related to particle/pore size distribution and competition between monomers and aggregates. As a matter of fact, a complete saturation is easier to reach with AbSolute HC material of narrow particles and pores distribution, and seems to be easier to reach at lower concentration. Table 6 shows the summary of those results at C/C0 0.1 (Q=10 %). Table 6: Summary result of dynamic binding capacity (Q) for the resins studied. MabSelect Sure resin Absolute HC resin IgG conc. Flow rate Q (g/ L resin) IgG conc. Flow rate Q (g/ L resin) (g/l) in feed (cm/h) at 10 % C/C0 (g/l) in feed (cm/h) at 10 % C/C As can be seen better results of dynamic binding capacity are obtained with AbSolute HC gel. The highest values are observed on Absolute HC with IgG at 300, 600 and 1000 cm/h with a range of ; and g/l depending on IgG concentration in feed at concentration at 0.13 g/l and 2.3 g/l. Nessa Publishers Page 10

11 Impact of speed on purity on AbSolute HC Beside the dynamic capacity at high flow rate, the ability of a Protein A affinity media to increase an antibody purity is obviously of paramount importance in such media performance evaluation. Table7, figures 4 and 5 show the results achieved in this phase to evaluate the impact of high velocities on Nimotuzumab purity and yield using different concentrations in the feed. The loading being the longest step in the process, increasing this step s speed will have a dramatic impact on the productivity. Three different linear velocities are tested for loading: 300 cm/h, 600 cm/h and 1000 cm/h. In some experiments, a higher speed is applied for either the wash step (Exp #4 and #9) or the elution step (Exp#5 and #10).As a matter of fact, increasing those steps speed will allow to decrease further the cycle time with some direct impact on production schedules if the method is implemented at manufacturing scale. Table 7: Summary table of speed impact on purity with Absolute HC Exp. Flow rate IgG Yield (%) Purity by Purity by No. (cm/h) conc. HPLC- SDSin feed SEC (%) PAGE (%) (g/l) ,23 97,05 94, ,49 97,53 95, ,06 96,22 95, cm/h: load, elution and CIP; 600 cm/h: wash 99,08 97,35 96, cm/h: load, wash and CIP; 600 cm/h elution 0,13 99,93 97,33 96, ,91 97,25 96, ,77 97,66 96, ,93 97,9 96, cm/h: load, elution and CIP; 600 cm/h: wash 96,78 97,63 95, cm/h: load, wash and CIP; 600 cm/h: elution. 2,3 98,6 97,7 95,14 Nessa Publishers Page 11

12 Figure 4: Pictures of reduced (right) and non-reduced SDS-PAGE (left) on some experiments. Figure 5: Chromatogram profile and purity result by HPLC-SEC. Name Retention Time Area % Area Height 1 Dimer mab Figure 5: Chromatogram profile and purity result by HPLC-SEC. As reported in table 5 and figure 4, high purity and recovery values are achieved for each condition evaluated using Absolute HC resin at both 0.13 and 2.3 g/l IgG concentration on feed (purity being measured by HPLC- SEC and SDS-Page electrophoresis). No significant loss of purity and yield are observed for speeds until 1000 cm/h for loading, and until 600 cm/h for wash and elution steps (the maximum linear velocity tested for those steps). Process-related impurities such as PA leakage and host cell protein (HCP) were also determined in these experiments. Figures 6 and 7 show the results obtained. Nessa Publishers Page 12

13 Figure 6: PA leakage results at different conditions tested with Absolute HC. For each condition, the left column shows the result achieved at 0.13 g/l feed concentration, and the right column at 2.3 g/l. Figure 7: HCP results at different conditions tested with Absolute HC. For each condition, the left column shows the result achieved at 0.13 g/l feed concentration, and the right column at 2.3 g/l. According to figures 6 and 7 the Protein A leakage is similar for all the tests performed with different velocities. There is no significant difference either in HCP values for eluted IgG under various operating conditions tested with the same concentration of IgG in feed solution. Some slight difference is observed for HCP content in the tests performed with higher feed concentration (2 g/l) :HCP is increased when a higher elution speed is used than loading and washing. However such HCP content in the elution stream is still within specifications typically defined for a Protein A capture step. Nessa Publishers Page 13

14 Lifetime study Cleaning, sanitization, and storage procedures that preserve resin function and integrity are essential for obtaining reasonable resin lifetimes. The behavior of the DBC and purity are evaluated on 200 cycles. Figures 8 shows the results. Figure 8: DBC on Absolute HC over 200 cycles. Factors that influence resin lifetime include the nature of the feed stream and raw materials used in its purification, the resistance of the resin to cleaning and sanitization agents, and proper storage conditions. During this test, less than 10 % decrease of 10 % BDC is measured after 200 cycles with 0.1M NaOH CIP solution every 10 cycles for 20 minutes contact time per cycle. Beside NaOH CIP every 10 cycles, a PAB hygienization step is applied as CIP at each cycle, with also 20 minutes as contact time. Purity and process-related impurities are also analyzed. Figures 9, 10 and 11 show the results. Figure 9: Purity results by HPLC-RP during 200 Cycles Nessa Publishers Page 14

15 Figure 10: Protein A leakage results during 200 cycles. Figure 11: HCP results during 200 cycles. During the lifetime test, no significant loss of purity is observed over the 200 cycles (figure 9). Protein A leakage in the elution is stable as well, and even though a slight increase in HCP content is observed, it is staying within the limits typically defined for this step for such attribute. Furthermore, as a final observation and as it is mentioned in the experimental section of this article, the eluted product is also analyzed between two NaOH CIP s, for cycles 31, 33, 35, 37 and 39 whose CIP step is carried out with the acidic PAB mixture only. No significant difference is observed regarding purity, HCP and protein A leakage. Nessa Publishers Page 15

16 Conclusions It was shown during the study described in this article that AbSolute HC Protein A media exhibits high Dynamic Binding Capacities at low and high speed for Nimotuzumab capture. Beside this property of higher loading, it was verified that product quality stays within specifications of purity, and is not impacted when the speed of loading step, washing step or elution step is increased. Both factors capacity and speed will allow to increase the productivity values to be reached with Protein A media, expressed in terms of quantity of Nimotuzumab produced per Liter of resin per unit of time. Based on those results, high performances in terms of productivity, purity, yield can be expected from an implementation at larger scale of this capture step, with some stable results over 200 cycles under the conditions tested in this study Nessa Publishers Page 16

17 References 1. AbSolute High Cap Revolutionary Protein A Media. Downstream Processing. BioProcess International Industry. Yearbook Dynamic binding capacity study on MabSelectSuRe LX for capturing high-titer monoclonal antibodies. GE Healthcare Life Sciences. Application note AA. 3. User Guide. AbSolute High Cap by Novasep High Performance Protein A Media. Revision 0: November Shukla A, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies. Application of platform approaches. J Chromatogr B. 2007; 848: Swinnen K, Krul A, Van Goidsenhoven I, Van Tichelt N, Roosen A, Van Houdt K. Performance comparison of protein A affinity resins for the purification of monoclonal antibodies. J Chromatogr B. 2007; 848: G.Guiochon, A. Felinger, D.G. Shirazi, A.M. Katti, Fundamentals of Preparative and Nonlinear Chromatography, 2 nd ed., Academic Press, San Diego, G. Guiochon, B. Lin, Modeling for Preparative Chromatography, Academic Press, New York, Candy K.S. Ng, Hector Osuna-Sanchez, Eric Valéry, Eva Sorensen, Daniel G. Bracewell. Design of high productivity antibody capture by Protein A chromatography using an integrated experimental and modeling approach. J Chromatogr B. 2012; 899: R. Hahn, P. Bauerhansl, K. Shimahara, C. Wizniewski, A. Tscheliessnig, A. Jungbauer, J. Chromatogr. A 1093 (2005) 98. Nessa Publishers Page 17