A midstream and downstream purification process at the 50 L scale for purification of a highly concentrated mAb is described
Subcutaneous (SC) administration of monoclonal antibodies (mAbs) has several benefits over intravenous (IV) infusion, including being less invasive, requiring shorter administration times, and the possibility for self-administration. However, to achieve the desired dosage within the volume limitations of SC administration, the formulation must be highly concentrated, often exceeding 100 g/L and sometimes reaching upwards of 200 g/L. Here we demonstrate, using only our products, how you can achieve a final mAb concentration of over 200 g/L whilst maintaining key quality attributes.
Introduction
Production of a highly concentrated mAb requires many process steps to work in harmony to deliver a suitable quality bulk drug substance. It may also require many products and pieces of equipment often from various vendors. However, this can cause difficulties due to different lead times for equipment and materials.
In this study, we ran a complete mAb purification process using only our products at a 50 L scale as outlined in Figure 1. A few steps were investigated to a greater extent, including:
- Running a viral filtration step using Pegasus™ Protect and Pegasus™ Prime virus filters in combination with ÄKTA pilot™ 600 chromatography system.
- Gentle and quick formulation of the mAb up to 50 g/L on traditional tangential flow filtration (TFF).
- Final concentration on a single-pass tangential flow filtration (SPTFF) step to reach a final concentration at 200 g/L.
- Final 0.2 µm filtration on Supor™ Prime sterilizing grade filter.
Fig 1. An overview of the mid- and downstream purification process.
Materials and methods
Cell culture
We ran a fed-batch culture using HyClone™ ActiPro™ cell culture media, HyClone™ Cell Boost™ 7a supplement, and HyClone™ Cell Boost™ 7b supplement at 50 L scale in an Xcellerex™ XDR-50 bioreactor to produce a monoclonal antibody.
Clarification using depth filters
Before harvest, we rinsed the Supracap™ 100 and Stax™ depth filter capsules with DI water and primed them with PBS. They were then drained to minimize product dilution.
We performed the coarse depth filtration for cell removal on a 1 m2 Stax™ PDK7 capsule followed by the fine depth filtration performed on 5 × PDCX 20 inch Supracap™ 100 filter capsules run in parallel (0.50 m2). The selected flux setpoint was 50 LMH for the primary depth filters and 100 LMH for the secondary depth filters. We ran the primary and secondary clarifications separately to allow for sampling and turbidity measurement in between.
After the depth filtration, we filtered the product through a Supor™ EKV 0.2 µm membrane (1500 cm2).
Affinity capture chromatography and viral inactivation
The clarified depth filtrates were pooled and captured on a 0.8 L ReadyToProcess™ MabSelect PrismA™ chromatography column on an ÄKTA pilot™ 600 chromatography system over 5 cycles. The load was set to 55 g/L. The chromatography method used is shown in Table 1 below.
Table 1. Capture MabSelect PrismA™ chromatography method| Step | Column volume (CV) | Buffer/material | Flow rate (residence time) |
|---|---|---|---|
| Equilibration | 3 | 20 mM sodium phosphate (NaP), 500 mM NaCl, pH 7.0 | 4 min |
| Load | N/A | Clarified harvest mAb | 6 min |
| Wash 1 | 1.5 | 20 mM NaP, 500 mM NaCl, pH 7.0 | 6 min |
| Wash 1 | 3.5 | 20 mM NaP, 500 mM NaCl, pH 7.0 | 4 min |
| Wash 2 | 1 | 50 mM sodium acetate, pH 5.5 | 4 min |
| Elution | 3 | 100 mM sodium acetate, pH 3.5 | 6 min |
| Strip | 2 | 100 mM acetic acid (HAc) | 4 min |
| Clean-in-place (CIP) | 3 | 500 mM NaOH | 5 min |
| Re-equilibration 1 | 1.5 | 20 mM NaP, 500 mM NaCl , pH 7.0 | 6 min |
| Re-equilibration 2 | 1.5 | 20 mM NaP, 500 mM NaCl, pH 7.0 | 4 min |
The eluate pool (at a titer of 34.7 g/L and 5.1 L) was viral inactivated for 60 min at pH 3.7 using 4 M acetic acid. We then adjusted the pH to 5.0 with 2 M Tris base and the product solution was filtered on a Supor™ EKV sterilizing grade filter, 0.2 µm, 1500 cm2. The product volume was 6.0 kg and had a titer of 29.1 g/L after viral inactivation and filtration.
Polishing chromatography on Capto™ S ImpAct resin
The viral inactivated (VI) mAb product was polished using a 0.8 L ReadyToProcess™ Capto™ S ImpAct chromatography column on an ÄKTA pilot™ 600 chromatography system over 4 cycles. The load was set to 70 g/L. The chromatography method used is shown in Table 2.
Table 2. Polishing method using Capto™ S ImpAct chromatography column| Step | Column volume (CV) | Buffer/material | Flow rate (residence time) |
|---|---|---|---|
| Equilibration | 5 | 50 mM sodium acetate, 50 mM NaCl, pH 5.0 | 5.4 min |
| Load | N/A | Adjusted VI pool, pH 5.0 ± 0.2 | 5.4 min |
| Wash | 5 | 50 mM sodium acetate, 50mM NaCl, pH 5.0 | 5.4 min |
| Elution | 7 | 50 mM sodium acetate, 200 mM NaCl, pH 5.0 | 5.4 min |
| Strip | 3 | 1 M NaCl | 5.4 min |
| CIP | 3 | 0.5 M NaOH | 5.4 min |
| Re-equilibration | 5 | 50 mM sodium acetate, 50 mM NaCl, pH 5.0 | 5.4 min |
Product conditioning and polishing chromatography on Mustang™ Q XT chromatography membrane capsules
The Capto™ S ImpAct chromatography resin eluate pool (7.8 kg at a titer of 21.3 g/L) was conditioned to pH 6.0 and a conductivity below 11 mS/cm using 100 mM sodium acetate, pH 8.3. The product weight was 40.9 kg with a titer of 4.1 g/L after conditioning.
A second polishing step was conducted on Mustang™ XT 5 chromatography membrane using an ÄKTA pure™ 150 chromatography system over 17 cycles in flowthrough mode. The load was set to 2000 g mAb/L. The chromatography method used is shown in Table 3.
Table 3. Polishing method using Mustang™ Q XT chromatography membrane| Step | Column volume (CV) | Buffer/material | Flow rate (MV/min) |
|---|---|---|---|
| Equilibration | 20 | 50 mM sodium acetate, pH 6.0 | 10 |
| Load | N/A | Adjusted Capto™ S ImpAct eluate | 10 |
| Wash | 10 | 50 mM sodium acetate, pH 6.0 | 10 |
| Strip | 5 | 1 M NaCl | 10 |
| CIP | 5 + 30 min hold | 1 M NaOH | 10 |
| Re-equilibration | 40 | 50 mM sodium acetate, pH 6.0 | 10 |
Virus filtration
The flowthrough eluate pool from Mustang™ Q XT chromatography membrane was 0.2 µm filtered using Supor™ EKV sterilizing grade filter 1500 cm2 (total weight of 41.7 kg and a titer of 4.0 g/L), and then applied to a filter train consisting of Pegasus™ Protect prefilter and Pegasus™ Prime virus filter connected to an ÄKTA pilot™ 600 chromatography system. We primed the filter train with the Mustang™ Q XT chromatography membrane equilibration buffer before we filtered the product.
The filtration was performed through a pressure-flow controlled UNICORN™ method with a maximum delta column pressure set at 2.5 bar (36.3 psi, 0.25 MPa). UNICORN™ software will not go over 80% of the pressure limit meaning that the maximum allowed pressure was 2.0 bar (0.20 MPa, 29 psi). The flow was set to 200 mL/min. The system maintained a steady delta filter pressure, and the flow did not decrease over time.
After the virus filtration, the product weight was 41.6 kg and we determined the titer to be 4.0 g/L. The UNICORN™ method used is shown in Table 4.
Table 4. Virus filtration method using Pegasus™ Protect- and Pegasus™ Prime virus filter| Step | Volume (mL) | Buffer | Flow rate (MV/min) |
|---|---|---|---|
| Equilibration | 300 | 50 mM sodium acetate, pH 6.0 | 200 |
| Load | N/A | Adjusted Capto™ S ImpAct resin eluate | 200 |
| Wash | 1000 | 50 mM sodium acetate, pH 6.0 | 200 |
Final formulation through TFF
Before starting the TFF step, we rinsed the 30 kDa Delta membrane of 0.5 m2 and integrity tested at a pressure of 4 bar (58 psi, 0.4 MPa) with a passing result. We performed membrane cleaning in place (CIP) using 0.25 M NaOH followed by a water flush to remove the CIP agent. A normalized water permeability (NWP) test showed a value of 201 LMH/bar (at 20°C). We then primed the membrane with formulation buffer.
During the initial concentration of the viral filtered product, the volume was kept at about 6 L in the reservoir of the ÄKTA™ flux 6 system. Once all the product had been transferred, we reduced the volume to about 2.89 L. The UF part of the TFF step took about 73 min with a transmembrane pressure (TMP) of 1.5 bar (21.8 psi, 0.15 MPa) and a recirculation feed flux of 360 LMH. The following DF was performed took 49 min with the same TMP and feed flux settings. An average permeate flux of 54.6 LMH was obtained reaching 7 DV.
After the DF, we opened the retentate valve completely, and the recirculation feed flux lowered to 120 LMH. We closed the permeate valve and the product was allowed to recirculate for 15 min to increase the recovery by gentle mixing and enhanced diffusion of mAb from the membrane surface back into the solution.
The final formulated mAb retentate was collected and the weight was 2844 g with a titer of 53.0 g/L and a total mAb content of 150.7 g. We performed two flushes of the ÄKTA™ flux 6 system with 450 mL of formulation buffer for each flush. The total flush was collected separately and weighed 1039 g with a titer of 12.18 g/L and a total mAb content of 12.7 g. The total recovered mAb amount was 163.4 g over the TFF step.
Both the retentate and the flush fraction were pooled with a weight of 3851 g, and a titer of 41.4 g/L. Before the final concentration on SPTFF, we filtered the product pool on a 0.2 µm filter.
The TFF membrane was cleaned in place after use and flushed with water. We performed a post-UF/DF NWP test, which gave a value of 202 LMH/bar (at 20°C), which showed full recovery of the membrane.
Figure 2 below shows the TMP and permeate flux during the UF and DF. Here we see that the permeate flux decreases as the product is concentrated during the UF.
The drop in flux and TMP after about 75 to 80 min was due to lowering the recirculation flow for rearranging the buffer vessel and permeate collection vessel before the DF started. Once the DF started, we see that the permeate flux increases initially, which may be an effect of the viscosity change due to the buffer exchange. The TMP is seen to be stable during the UF and DF.
Fig 2. TMP and permeate flux during the UF and DF of the TFF step.
Final concentration through SPTFF
For the final concentration on a single-pass TFF unit (SPTFF), we assembled seven individual Delta 30 kDa, 93 cm2 membranes with a Cadence™ single-pass modular kit into a 4-in-series SPTFF unit. We ran the step using a Quattroflow 150S pump. We controlled the retentate flow through a valve preventing unstable performance, which most likely could lead to a too high concentration as the mAb concentration increases over the SPTFF unit. Another prerequisite was to start with a lower product concentration, and this was why we did not concentrate the mAb solution above 50 g/L in the previous TFF step.
Before starting the process, the assembled membranes in the 4-in-series SPTFF unit were cleaned in place with 0.25 M NaOH, flushed with water, and integrity tested at 4.0 bar (with a passed result and the following NWP test gave a value of 202 LMH/bar at 25°C). The hold-up weight in the system was 44.8 g.
We carried out an initial flux excursion and used a previous optimization of our TFF and SPTFF step as guidance. The optimal setting was finally found at a feed flow of about 15 to 17 LMH, feed pressure of 2.8 bar (40.6 psi, 0.28 MPa) and retentate pressure of 2.0 bar (29.0 psi, 0.20 MPa). The volumetric concentration factor then became about 4.7 on average. These parameters were used for the SPTFF step. However, at these settings, we only reached a titer of 180 g/L, and we decided to reprocess the product on the SPTFF step.
We diluted the product with formulation buffer to a titer of 42.7 g/L, and the starting product weight became 3473 g. The SPTFF membranes were cleaned and a new NWP test was done with a result of 207 LMH/bar (at 25°C). During the flux excursion, we found that a feed flow of about 11 to12 LMH, feed pressure of 3.2 bar, and retentate pressure of 2.05 bar (29.7 psi, 0.21 MPa) gave a higher volumetric concentration factor than previously. This time, the volumetric concentration factor became about 5.4 on average.
The SPTFF final concentration step took 4 h and 40 min at steady pressures. At the end, we performed three separate flushes with about 45 g of buffer for each flush. The titer in the concentrated mAb was 225.6 g/L after the SPTFF step.
After the process, the SPTFF unit was cleaned in place and the following NWP test gave a value of 199 LMH/bar (at 25°C), showing full recovery of the membranes in the SPTFF unit.
Figure 3 below shows the accumulated retentate- and permeate weights throughout the SPTFF process, as well as the feed- and retentate pressure for run 2.
Fig 3. Accumulated retentate- and permeate weights throughout the SPTFF step, as well as the feed- and retentate pressure.
Filtration using Supor™ Prime membrane of the final high-concentration mAb product
We filtered the final mAb product, with a concentration of 225.6 g/L, through 0.2 µm Supor™ Prime sterilizing grade filters.
The weight of the final product was approximately 627 g. We have previously determined the filter capacity to be 390 L/m2 when filtering at a flux of 400 LMH at a max. pressure setting of 2.0 bar (0.20 MPa, 29 psi) on a 3 cm2 filter. However, for the product solution we had, a 3 cm2 would not be sufficient, and we therefore filtered the product on a 240 cm2 Supor™ Prime filter despite having an overcapacity.
The filtration was quick and took 10 min performed at a constant pressure of 0.75 bar (10.87 psi, 0.075 MPa). The final product had a titer of 240.3 g/L, which is slightly high and shows the difficulty with measuring titer on highly concentrated mAb solutions.
Results and discussion
Cell culture
The cell culture used for this study was a fed-batch CHO cell culture. At the point of harvest, the viability was at 95%, the mAb titer was 5.1 mg/mL, the maximum cell density reached was 51 × 106 cells/mL, and the turbidity at harvest was 3020 FNU.
Clarification
The depth filtration on the primary Stax™ PDK7 filter and the secondary Supracap™ PDCX filter went as expected. Both grades are slightly tighter than their Stax™ mAx counterparts and offer improved fine particle removal from this challenging high-cell-density feed stream.
The turbidity from the depth filtration, is shown in Figure 4. As expected, the tighter depth filter train of Stax™ PDK7 and Supracap™ PDCX generated a filtrate pool with a low turbidity (less than 10 NTU).
Fig 4. Turbidity after depth filtration and Supor™ EKV filtrations.
Affinity capture chromatography, viral inactivation, and polishing chromatography
The capture chromatography using MabSelect PrismA™ resin, as well as both polishing steps on Capto™ S ImpAct resin, and Mustang™ Q XT membrane performed as expected. This demonstrates the suitability of running Mustang™ Q XT membranes on an ÄKTA pure™ 150 system during process development. An example of the chromatograms is shown in Figure 5.
The viral inactivation step at low pH also performed as expected. It is not uncommon to see a slight increase in product cloudiness when increasing the pH after the low pH viral inactivation. A depth filtration step is typically employed after the viral inactivation to remove the precipitated impurities. In this case, we managed to filter directly using a 0.2 µm Supor™ Prime filter 1500 cm2 and the differential pressure during the filtration was below 0.1 bar (0.01 MPa, 1.5 psi).
Fig 5. Chromatograms from (A) MabSelect Prisma™ chromatography resin, (B) Capto™ S ImpAct chromatography resin, and (C) Mustang™ Q XT chromatography membrane capsules.
Virus filtration
A virus filtration step is traditionally performed at constant pressure, that is, through pressurizing a stainless-steel vessel with the product solution being transferred through tubing into the virus filter. However, we have shown that virus filtration can be performed using an ÄKTA pilot™ 600 chromatography system with a filter train consisting of Pegasus™ Protect prefilter and Pegasus™ Prime virus filter.
The chromatogram from the virus filtration is shown in Figure 6. It shows how the ÄKTA pilot™ 600 chromatography system with a UNICORN™ software method can maintain a steady delta column pressure throughout the virus filtration and adjust the flow rate accordingly. In this case, we could not see any flow decay during the virus filtration.
Fig 6. Chromatograms from the virus filtration on ÄKTA pilot™ 600 chromatography system.
Final TFF formulation
During the final formulation we showed that the mAb solution could be quickly formulated on T-series cassettes with Delta membrane of 30 kDa through both UF and DF using an ÄKTA flux™ 6 chromatography system. We also showed that keeping the concentration at 50 g/L resulted in a gentle processing and minimized the increase in aggregation over the step.
Final SPTFF concentration
Following the TFF concentration we ran the SPTFF step twice and for the last run, we made a final concentration up to 225.6 g/L using a stand-alone SPTFF operation with a 4-in-series SPTFF unit with a 30 kDa MWCO Delta membrane. We demonstrated a concentration factor of 5.4 at steady feed and retentate pressures throughout the entire process step. However, we could see that the aggregation level increased over this step, which was expected as we ran the SPTFF step twice. However, the level of aggregation in the final SPTFF retentate seems to be dependent on the feed pressure, hold-up time in the unit, and concentration factor to reach, but may also depend on the starting level and start titer.
Supor™ Prime sterile filtration
The final concentrated product was filtered through a 0.2 µm Supor™ Prime filter. With the previously determined throughput capacity of 390 L/m2 we could filter all the product quickly without a pressure increase.
Analytical results
The mAb production process was monitored using several different analytical methods. Analytical results are listed in Table 5 for the final product, and Table 6 contains step yields and aggregate levels after each process step. The step yields were as expected, and the level of aggregates was low throughout the process. A significant increase was seen over the SPTFF step, though this was expected as the formulation buffer had not been optimized. However, the level of aggregation over the SPTFF step may be kept lower by running the step at lower pressures, lower hold up time in the SPTFF unit, and by concentrating to a slightly lower titer as well.
Charge variants were monitored over the process, but the distribution of charge variants did not change significantly over the process.
Table 5. Analytical results for the final bulk product| Assay | Result | Acceptance criteria |
|---|---|---|
| HCP (ng/mg drug product) | 15.2 | < 100 |
| DNA (ng/mL) | < 0.001 | < 10 ng/dose for mAbs US FDA |
| Aggregates (%) | 3.3 | mAb dependent |
| Charge variants (% main) | 40 | Consistent profile |
| Concentration (g/L) | 240 | Not available |
| Protein A (ng/mg drug product) | < 0.5 | Not available |
The step yields in Table 6 are as expected, but slightly low for the MabSelect PrismA™ step. However, this is most likely higher, and the stated value of 85.3% may be an effect of measuring the titer with a Cedex bioanalyzer for the load, but using a spectrophotometer for the eluate—analytical variations will occur due to different methods for measuring.
The step yield for the Supor™ Prime filtration on the final product is most likely a false value, as it is somewhat difficult to pipette a viscous high-concentration mAb.
Table 6. Step yields (%) and aggregate level after each step| Process step | Step yield (%) | Aggregate level (%) |
|---|---|---|
| Depth filtration | 90.6 | N/A |
| MabSelect PrismA™ resin chromatography | 85.3 | 0.5 |
| Viral inactivation and Supor™ EKV filtration | 97.4 | 0.7 |
| Capto™ S ImpAct resin chromatography | 95.6 | 0.7 |
| Mustang™ Q XT membrane chromatography | 100.2 | 0.7 |
| Viral filtration | 98.6 | 0.8 |
| TFF formulation | 97.7 | 0.7 |
| SPTFF concentration | 97.7 and 98.9 |
1.4 (run 1) and 3.0 (run 2) |
| Supor™ Prime sterilizing grade filtration | 108.7 |
3.3 |
Conclusions
In conclusion, we have shown a complete mAb production process was run demonstrating the interconnection of our products. In addition, we have shown:
- Depth filter train Stax™ PDK7 plus Supracap™ PDCX option offers a filtrate with very low turbidity.
- Successful running of Mustang™ Q XT chromatography membrane capsules on an ÄKTA pure™ 150 chromatography system.
- Successful running of virus filtration using Pegasus Protect and Pegasus Prime as a filter train connected on an ÄKTA pilot™ 600 chromatography system. The system kept constant pressure by controlling the flow rate over time.
- Fast and gentle formulation of mAb using an ÄKTA flux™ 6 system in combination with a T-series cassette with a 30 kDa MWCO Delta regenerated cellulose membrane, 0.5 m2.
- Final concentration to above 200 g/L on an SPTFF unit with a concentration factor above 5 achieved.
- The level of aggregate at 3.3% is slightly high for a mAb solution, but expected given that we performed no optimization of the formulation buffer.
- The final filtration on Supor™ Prime membrane was successful and showed a high capacity for high-concentration mAb solutions at 390 L/m2.
CY46012-16Jun25-AN