Transferring optimized purification development results to pilot- and full-process scale is a key component for successful MAb production. Scale-up needs to be robust, predictable, and the optimized conditions for removing aggregates, HCP, DNA, and viruses must be maintained or, preferably, improved. The addition of readyto-use solutions to the upstream/downstream process significantly shortens the time-to-clinic journey for MAbs.
Scaling up with AxiChrom columns
In the scale-up described, the conditions optimized on 4.7 ml HiScreen™ prepacked columns were transferred to a large-scale, two-step purification process run using ÄKTApilot™ on stainless steel AxiChrom 70/300 columns. This process employed MabSelect SuRe™ and Capto™ adhere, which were packed automatically to bed heights of 20.5 cm and 14.1 cm, respectively. A DoE approach and Monte Carlo simulation were used to verify the robustness of the scaled-up process.
Large-scale NFF and CFF steps were included in the overall purification flow scheme. These filtration steps were also optimized by determining robust parameters that consistently produced high yields.
Target MAb was expressed in CHO cells cultured in a 120 l stirred-tank steel bioreactor with a working volume of 100 l. After harvesting the cells by centrifugation, the supernatant was filtered.
Factorial design was used to optimize the MabSelect SuRe step. Set criteria were a yield of more than 90%, an aggregate content of less than 15%, and an HCP content of less than 70 ppm. Results showed that these criteria were met with a pH of 3.65 to 3.90 and a load of 17 to 34 g/l. As the most important factor of capture was judged to be monomer yield, a Monte Carlo simulation was set up to investigate this parameter. The simulation had initial ranges: load 24 to 27.5 g/l, NaCl concentration in wash 430 to 470 mM, elution flow rate 140 to 160 cm/h, elution pH 3.6 to 3.8.
The simulation predicted a yield of 92.4 to 95.6% (Fig 1A). These results were then optimized in silico by increasing the load range to 25.7 to 30.0 g/l and decreasing pH to 3.5 to 3.7. A new simulation gave an improved yield within a narrower range (94.7% to 96.0%), thereby improving the robustness of the step (Fig 1B).
The average yield of scaled-up capture runs in the AxiChrom 70/300 column was 96.2%. The incidence of protein aggregates in the sample was 12% and HCP content was reduced from approximately 36 000 to 24 000 ppm. Figure 2 shows overlaid chromatograms from the first three cycles with identical loads.
A sweet-spot plot was set up for monomer purity more than 99%, a yield greater than 85% plus negligible levels of HCP and ligand leakage. The result showed a large operating space in which the predefined criteria were met, suggesting that it was feasible to achieve the desired purity and yield at a starting aggregate concentration of 9% to 12.5%.
Monte Carlo simulation results indicated a purity of 99.2% to 100.1% (equivalent to an aggregate content of 0.0% to 0.8%) and a monomer yield of 82.5 to 86.7%. Analytical gel filtration data showed that the scaled-up process reduced the starting aggregate concentration of 12% to 0.6% in a single step. The 86% monomer yield is considered good for a starting sample containing such a high level of aggregates.
The final formulation step that follows polishing on Capto adhere comprised diafiltration at a 6-fold concentration followed by continuous diafiltration with seven volumes of formulation buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0) and one last concentration. This process was highly efficient, cutting the filtration time to less than 1 h and reducing buffer volumes.
The scaled-up protocol was thus deemed successful; the two-step MAb process had been run on AxiChrom columns at pilot scale in a predictable and robust manner with all preset purification criteria maintained.
Traditional vs. ready-to-use solutions
The key final goal of this study was to compare the scaled-up, two-step MAb process based on a 100 l cell culture in a traditional stainless steel bioreactor and purification in AxiChrom columns with one run on ready-to-use products from the ReadyToProcess platform.
The ready-to-use process equipment used included cell culture in WAVE Bioreactor™ System 200, filtration with ReadyCircuit™ fluid management assemblies, and purification with ÄKTA™ready system and ReadyToProcess columns prepacked with MabSelect SuRe and Capto adhere.
From a quality point of view, no difference between the traditional and ready-to-use processes could be demonstrated. In both cases, aggregates were reduced from around 10% to 0.5% to give a monomer yield of 81%. The respective final MAb products contained the same low amount of impurities and total yields were the same. Compare also the consistency of the MabSelect SuRe capture step run on the ReadyToProcess format (Fig 3) with that run on the conventional AxiChrom column (Fig 2).
Improved process time with ReadyToProcess solutions
Significant time was saved using the ready-to-use process. The time saving originated from the buffer exchange step (42% time reduction), chromatography steps (58% time reduction), and formulation step (31% time decrease). Although the actual run time for filtration using the traditional format was shorter (the flux of the hollow-fiber format is lower than a cassette), faster set-up times meant that the ReadyToProcess format was still quicker, even for filtration.
Comparing total process times revealed that the ready-to-use process was completed in just 23 h while the traditional process required 45.5 h (Table 1). This almost 50% reduction was due the ready-to-use format eliminating several labor-intensive steps, for example, column packing and validation. In addition, WAVE Bioreactor System 200 eliminated upstream cleaning and cleaning validation, which saved even more time. Presterilized, disposable bioreactor bags instead of traditional stainless steel vessels also helped increase product safety. The use of new ReadyCircuit fluidprocessing assemblies in the fluid management and filtration steps, as well as the use of a disposable flow path with ÄKTA ready system significantly contributed to the observed time reduction.
After PreDictor plate screening and optimization with HiScreen prepacked columns, a scaled-up protocol was developed with the help of DoE and Monte Carlo simulation. This simulation proved to be highly robust when transferred to AxiChrom columns run on ÄKTApilot system. Finally, running the upstream cell culture and downstream purification on equipment from the ReadyToProcess platform cut processing times by 50% compared with traditional process equipment, while maintaining the same high quality criteria of the purified final product.
For further information, see the following application notes:
· A flexible antibody purification process based on ReadyToProcess products, Code no. 28-9403-48
This article is an extract from Upstream& Downstream (Vol 3) pp.14–16 (2010) GE Healthcare 28-9863-33AA.