Material and methods

Materials

Three prepacked 1 mL HiTrapTM DEAE FF columns were used for the capture step, and a 5 mL HiTrapTM Phenyl FF (high sub) column was used in the polishing step (all from Cytiva, Uppsala, Sweden). The buffers in each step were the same as those used in our previous study (Löfgren et al., 2018), and the chromatography columns were sanitized with 1 M NaOH after each cycle. Conditioning between the two steps was achieved with inline dilution using a high-salt concentration buffer as a conditioning buffer and a 1.4 mL dynamic mixer. The conductivity and pH of the conditioning buffer were adjusted to obtain the desired values after inline dilution, thus ensuring product binding on the polishing column. The ratio of eluate volume from the capture column to conditioning buffer volume was 1:1.6.
The continuous virus inactivation reactor was a Cytiva C-type column packed with 150-250 µm silica beads, with an inner diameter of 16 mm and a maximum height of 400 mm. The clarified supernatant was diluted inline at a ratio of 9:1 with a solvent/detergent mixture containing 10 vol% Polysorbate 20 and 3 vol% Tri-n-butyl phosphate, which was loaded from a 50 mL SuperloopTM (Cytiva). A 1.4 mL dynamic mixer was used for inline dilution of the supernatant. Cold water was pumped continuously through a cooling jacket to minimize the risk of microbial growth in the reactor, as this could plug the reactor. In addition, the reactor was sanitized with 1 M NaOH before the start of each run, and was filled with 20 vol% ethanol for storage.
The integrated continuous downstream process was implemented in two ÄKTATM pure 150 systems, controlled by the software UNICORNTM 7, (all from Cytiva, Uppsala, Sweden). One ÄKTA system was used for virus inactivation and the capture step with the PCC operation, and the other ÄKTA system was used for the polishing step. Each ÄKTA system included the following elements: two gradient pumps (pump A and B), a sample pump, several versatile valves (VV) to adjust the flow path, several column and inlet valves, an outlet valve, a fractionator to sample the product, and sensors for measuring UV light absorbance, conductivity, and pH.
An AgilentTM 1260 Infinity II HPLC system (Agilent Technologies Inc., Kista, Sweden) was used for the analytical experiments. The column used for concentration measurements was a PorosTM 50 HE column (Thermo Fisher Scientific Inc., Stockholm, Sweden) and a ZorbaxTM 300SB-C3 column (Agilent Technologies Inc) for purity.

Process setup

The setup was an adaptation of that used previously (Gomis-Fons, Andersson, & Nilsson, 2020); the main difference being the inclusion of continuous virus inactivation with solvent/detergent before the capture step (see Figure 2). In this setup, the capture columns used in the PCC operation were loaded directly from the virus inactivation reactor (red line in Figure 2). The recovery phases were performed with the sample pump (dotted blue line in Figure 2). The eluate from the third capture column was passed through a number of sensors (to measure UV absorption, conductivity, and pH) and was then loaded directly onto the polishing column, without a hold-up volume. Two outlet valves allowed the collection of the product in different parts of the downstream process for quantification of the productivity and the yield.
Two pumps were necessary for continuous virus inactivation: one to load the supernatant solution (pump AP), and another to dilute the supernatant inline with the solvent/detergent mixture (pump BP). Pumps AP and BP were also used to vary the load concentration by setting different proportions of the flow rate provided by the two pumps. Case c0 was run with detergent, and Case c1A was run with water. The reason why water, instead of detergent, was used in Case c1A was that the load concentration could not be changed in the current setup at the same time as inline dilution with detergent, since pump BP was used for both purposes. Thus, in Case c1A, pump BP was used to dilute the supernatant with water to adjust the load concentration, and in Case c0, pump BP was used to inject the detergent and mix it with the supernatant.

Analytical methods

A breakthrough curve experiment was performed with an ÄKTA pure 150 system. The supernatant was mixed inline with water at a ratio of 9:1 to emulate the loading conditions in the capture step, and then loaded onto the capture column. The outlet stream was sampled with a fractionator with a sample size of 2 mL. The breakthrough samples, together with a sample of the clarified supernatant, were then analyzed on the HPLC system. For the concentration measurements, aqueous buffers with different salt concentrations were used at a flow rate of 0.5 mL/min. The absorbance at a detection wavelength of 280 nm was used to determine the concentration, by using an extinction coefficient of 1.33 mL-1 mg cm-1, knowing that the path length of the cell was 0.2 cm. Resin utilization, defined as the amount of adsorbed product divided by the maximum amount of adsorbed product at a specific load concentration, was estimated from the breakthrough curve.
A U9-M UV monitor, included in each ÄKTA system, was used to obtain the amount of purified product in each cycle (hereinafter referred to as “product output”). The absorbance at a detection wavelength of 280 nm was measured and the concentration of the final product, which corresponds to the eluate from the polishing step, was then determined by using the extinction coefficient. The product output was calculated as the concentration multiplied by the eluate volume, which was obtained by multiplying the elution flow rate by the pooling time. The product output was used for the loading control, and to calculate the yield, defined as the product output divided by the amount of product loaded; the productivity, defined as the mass of product output divided by the process time and the total amount of resin in the process; and the specific buffer consumption, defined as the total amount of buffer consumed divided by the product output.
The breakthrough of both the capture and the polishing columns were collected during the run. The eluate from the polishing step was also collected during the whole run, while the eluate from the capture column was only sampled in the fourth cycle, when steady state had been achieved. Reversed-phase chromatography was performed to determine the purity of all these samples. Two buffers with different contents of water and acetonitrile were used (one as equilibration and loading buffer with 5 vol% acetonitrile, and another as elution buffer with 95 vol% acetonitrile) at a flow rate of 1 mL min-1. The absorbance at a detection wavelength of 280 nm was used to determine the concentration. The purity was then calculated by dividing the area under the peaks corresponding to the pure product by the total area under the peaks.
An ÄKTATM pure 150 system was used to validate the design of the continuous virus inactivation reactor by obtaining a residence time distribution curve. A U9-M UV monitor, included in the same ÄKTA system, was used to detect the product in the outlet of the reactor at a wavelength of 280 nm.

Process design

PCC design

The design of the PCC operation was based on the approach described by Godawat et al. (2012), in which several equations corresponding to a continuity constraint and several yield constraints were used to determine the PCC cycle times with the aid of an empirical model of a breakthrough curve. This approach is only valid for a specific flow rate, and different breakthrough curves must thus be obtained at different flow rates. For this reason, the loading flow rate was determined beforehand, and an experimental breakthrough curve was then obtained for this specific flow rate.
The flow rate was calculated by dividing the load volume by the minimum cycle time. The load concentration and volume were 0.74 mg mL-1 and 27 mL, respectively, as in our previous implementation of the process (Löfgren et al., 2018). The cycle time must be equal to, or longer than, the recovery time, based on the continuity constraint (Godawat et al., 2012). In this case, the capture and polishing steps were run simultaneously, and therefore the minimum cycle time was determined by the longest recovery time of these steps, as described previously (Gomis-Fons, Andersson, et al., 2020). By determining the flow rate in this way, at least the same yield and resin utilization as we obtained previously could be obtained at the minimum cycle time (Löfgren et al., 2018). The minimum cycle time was 79 min (the recovery times for the capture and polishing steps were 61 and 79 min, respectively), which led to a flow rate of 0.34 mL min-1. As the supernatant was mixed with detergent at a ratio of 9:1, the total incoming flow rate was 0.38 mL min-1. If the downstream process were to be connected to a perfusion bioreactor, the load flow rate and concentration would be determined by the harvest stream of the bioreactor, and would therefore be variable. For this reason, the values of flow rate and concentration given above should be considered as nominal design values, and they would correspond to the expected steady-state values from the bioreactor. Therefore, connection to a perfusion bioreactor would be possible by scaling the columns in the downstream process up or down to obtain the desired flow rate and resin volume.
The load volume was then optimized with the breakthrough curve in order to maximize resin utilization and obtain a high yield. Godawat et al. (2012) proposed a method based on the area under the breakthrough curve in the loading phase, in which two columns are interconnected to avoid any loss of product in the breakthrough. The area under the breakthrough curve of the first interconnected column, which corresponds to the amount of product breaking through this column and loaded on the second interconnected column, must be smaller than the dynamic binding capacity, to avoid product breakthrough in the second column. The design in the present study was more flexible, allowing this area to be greater than the dynamic binding capacity, thus some product loss was tolerated in the breakthrough. The concept for the design of the PCC operation based on a breakthrough curve is presented in Figure 3.
When a PCC cycle starts, the first column is already loaded with product from the previous cycle, corresponding to a load volume \(V_{1}\). At the end of a PCC cycle, the total loaded volume in a column is\(V_{2}\). The load volume in a cycle (hereinafter referred to as\(V_{\text{cycle}}\) or “cycle load volume”) is therefore equal to\(V_{2}-V_{1}\). The area under the curve between \(V_{1}\) and\(V_{2}\) (\(k_{1}\)) corresponds to the amount of product loaded onto the second column. In other words, the breakthrough of the first column during \(V_{\text{cycle}}\) corresponds to a load volume on the second column of \(V_{1}\). The area under the curve during\(V_{\text{cycle}}\) (\(k_{1}\)) must therefore be equal to the rectangle area between zero and \(V_{1}\), which is equivalent to\({k_{2}+k}_{3}\). The area under the curve between zero and \(V_{1}\)(\(k_{2}\)) is the product loss due to breakthrough, and was used to predict the yield by dividing the amount of product adsorbed (\(k_{3}+k_{4}\)) by the total amount loaded in the column (\(k_{2}+k_{3}+k_{4}\)). The resin utilization was estimated by dividing the amount of product adsorbed (\(k_{3}+k_{4}\)) in the column by the total binding capacity, estimated as\(k_{3}+k_{4}+k_{5}\).
It can be seen from Figure 3 that increasing the load volume (\(V_{2}\)) increases the resin utilization, but the yield falls as the result of increased product loss at breakthrough in the second column (\(k_{2}\)). There is thus a relationship between resin utilization, yield and load volume, as shown in Figure 4. The resin utilization remains above 70% for load volumes greater than 35 mL, while the yield is not significantly affected until load volumes of 60-70 mL are reached. In other words, the resin utilization can be significantly increased by allowing some product loss at breakthrough, as opposed to the approach described by Godawat et al. (2012), in which no product loss is allowed and the yield is therefore set at 100%.
The loss of binding capacity over time, due to column degradation, must be taken into consideration in the design of a chromatography process. An empirical model previously used by Godawat et al. (2012) was used to predict new breakthrough curves with reduced capacities corresponding to 80 and 90% of the original capacity: