Evaluation of Crossflow Filtration System

In an approach similar to option 2, in figure 5, the last three productions used an MF approach for cell harvest using a crossflow filtration system. The ad- vantage of this method is that it should theoretically eliminate the need for centrifugation entirely, thus speeding up the harvesting process for large vol- ume bioreactor runs. There were however issues with the current set up, namely fouling of the MF membrane, and precipitation of the protein after UF.

BR5 was used to evaluate the efficiency of the crossflow filtration system.

Samples were taken at each step for both NanoDrop™ readings and for band quantification. Due to the presence of other substances prior to purification, band quantification values are more representative of the protein concentra- tion. Total protein was calculated using a peak area vs. protein concentration standard curve (R²=0.9973) as described in section 2.7.2.

Protein content of samples from downstream processing are shown in figure 9. Figure 10 shows the visual appearance of the retentate and permeate after UF as well as the final protein in storage buffer. Band quantified protein con- tent and UV-vis spectroscopy readings are further visualized in figure 11, also

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displaying the full sequence of events in the downstream processing. The first step, MF, resulted in a small but significant loss of approximately 200-400 mg of protein. The readings are not accurate enough to determine the precise amounts of protein lost at each step, but for the sake of speculation, the dif- ference in protein before and after each step, as well as the protein found in the side streams is presented in table 6. These values were then averaged to get an approximate for the amount of protein lost. These values indicate that the ultrafiltration step is where most of the protein was lost.

Table 6 | Approximate protein lost in microfiltration and ultrafiltration steps.

Protein quantity dif- ference before and after (mg)*

Protein in side streams (mg)*

Approximated protein lost (mg) Microfiltration 215 ± 939 306 ± 115 260

Ultrafiltration 534 ± 893 287 ± 208.5 410

* Protein quantity values were derived from band quantification values done with three technical replicate meas- urements of the same SDS-PAGE band. Values are from the downstream processing of bioreactor run 5 (BR5) (n=1).

Figure 9 | Protein content of downstream processing samples. MF: microfil- tration; UF: Ultrafiltration (5000 Da pore size). MF Retentate: contains cells and some media; MF filtrate: contains media and proteins; UF concentrate:

contains concentrated protein; UF permeate, media and particles smaller than 5000 Da; UF wash 1: water used to wash the retentate side of the sys- tem; UF wash 2: water used to wash the permeate side of the system. Main samples are in blue, while side streams are in yellow. Protein quantity values were derived from band quantification values done with three technical rep- licate measurements of the same SDS-PAGE band. Values are from the downstream processing of bioreactor run 5 (BR5) (n=1).

MF membrane fouling is a commonly encountered issue [65] [70] [71]. In these productions, the first harvest using microfiltration worked sufficiently, but subsequent runs quickly clogged up the membrane and significant

0 200 400 600 800 1000 1200 1400 1600 1800 2000

After harvesting

MF retentate

MF filtrate UF concentrate

UF permeate

UF wash 1

Total protein (mg)

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cleaning issues were experienced. While this issue was overcome by first cen- trifuging the cells, this entirely defeats the purpose of replacing the centrifu- gation step with microfiltration. To avoid fouling, it is critical to operate the system at below critical flux, that is the maximum permeate flux that the crossflow system can sustain before it can no longer clear away cell debris from the membrane faster than the permeate flow brings new debris to the membrane surface [65]. According to van Reiss and Zydney [71], this issue can be easily fixed by operating the system at constant flux rather than con- stant transmembrane pressure. Some cutting-edge technologies also exist that try to minimize membrane fouling using improved fluid mechanics, such as vibrating membrane filtration for HCDF yeast culture [72], but these tend to have limited availability [65].

There were also major difficulties with the UF step. A significant amount of protein was lost, and both the UF retentate and the purified protein were un- usually cloudy (Figure 10A & 10C). The pH was measured as 13.62, indicating the presence of excessive amounts of salts. Since no similar issues were found in literature, the most likely explanation is salt precipitation of the protein due to the presence of sodium hydroxide in the solution. It was speculated that this sodium hydroxide was released from the UF membrane during con- centration of the protein, which could explain the large protein loss during this step (table 6). In this particular case, the age and previous use cases of the UF membrane were unknown. Therefore, a likely fix for this issue is to use an entirely new membrane in future productions. After harvesting, the purification and buffer exchange steps were done the same way as previously.

Figure 10 | BR5 down- stream processing sam- ples. A) Concentrated protein from BR5 (re- tentate); B) permeate from UF concentration of BR5; C) Final pro- tein. Protein was cloudy and had an ex- cessively high pH.

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Figure 11 | Protein content of downstream processing samples. MF: microfil- tration; UF: Ultrafiltration (5000 Da pore size). Before purification, band quantification gives the most accurate readings for protein content. After pu- rification, UV-Vis spectrophotometry (NanoDrop™) can be used since impu- rities have been removed. Band quantification was done with three technical replicate measurements of the same SDS-PAGE band. Nanodrop values were measured using UV-Vis spectrophotometry with 7-10 technical replicates. Val- ues are from the downstream processing of bioreactor run 5 (BR5) (n=1).

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