In this application note, we describe the use of AcroPrep™ 24-well cell clarification and sterile filtration plates (AcroPrep™ CCSF plates) for the efficient clarification and filtration of cell cultures and lysates from smaller organisms, including Saccharomyces cerevisiae and Escherichia coli. We discuss the preparation, processing, and analysis of the cultures and lysates, demonstrating significant reduction in turbidity and OD600 measurements, with protein recovery rates exceeding 88%. Our article concludes that the AcroPrep™ CCSF plates are effective for both yeast and bacterial cultures, offering a broad application range for cell clearance and lysate filtration.
Introduction
Overexpression of proteins has enabled access to highly purified proteins in large quantities for research and increasingly for medical purposes. Expression hosts can be mammalian cell lines like CHO or HEK293, but other organisms like yeast and bacteria are also commonly used.
The first purification step depends on whether the overexpressed protein is secreted into the media or stays inside the cell. For excreted proteins, the initial step is to remove the expression host cells from the crude culture. We can do this by centrifuging the mixture to sediment the cells, then collecting the supernatant for further processing. Additional smaller debris still present in the cleared supernatant can be removed in a subsequent filtration step, which can also sterile filter the media. For proteins expressed inside cells, cell disruption (breaking) or lysis is performed as an initial step to access the target protein, then we remove remaining cellular debris from the lysate, typically by filtration.
Overall, the clarification process described may be complex, particularly in early discovery phases involving multiple cultures with relatively small culture volumes. Our AcroPrep™ 24-well cell clarification and sterile filtration plates, fitted with a Seitz depth filter over a Supor™ EKV 0.65/0.2 µm membrane, are optimal for clarification of mammalian cell cultures such as CHO-cells, combining cell clearance and sterile filtration functionality in a single solution.
The mammalian cells removed by the plates measured approximately 15 to 30 μm in size. In this article, we describe the use of AcroPrep™ CCSF plates for clarification of cell cultures and lysates from smaller organisms, including bacteria (0.2 to 2 μm) and yeast (5 to 10 μm), processed by vacuum or centrifugation. The plates resulted in decreased cellular content and debris in the cultures and lysates, as demonstrated by reduced turbidity and OD600 absorbance measurements. Protein recovery rates exceeded 88%.
Materials and methods
Cell culture and lysate preparation
We grew a cell culture of S. cerevisiae (Brewer’s yeast) under sterile conditions in yeast extract peptone dextrose (YPD) broth at 30°C, shaking at 200 rpm after inoculation with a 1:100 dilution of preculture. We grew the culture overnight for 16 h to a final absorbance at 600 nm (OD600) of 33.0 to 34.0. Fresh cell culture was used for clarification with the AcroPrep™ CCSF plates.
Additionally, we lysed a part of the S. cerevisiae culture. To accomplish this, we first pelleted the S. cerevisiae cells by centrifugation at 1000 × g for 5 to 10 min. Then we discarded the supernatant by slow decanting and replaced it with an identical volume lysing buffer consisting of Zymolyase 20T at 2 mg/mL in 1 M sorbitol, 0.1 M EDTA, pH 7.4 to 8.0 (Y1 buffer), supplemented with 20 mM DTT just prior to use. After resuspending in the buffer, we incubated the cells in a ThermoMixer C instrument for 30 min at 35°C under shaking.
We homogenized the solution for 45 s using 3 mm beads at a frequency of 30 Hz. We then grew a culture of a clone of E. coli strain TOP10 transformed with plasmid pUC19, under sterile conditions in tryptic soy broth (TSB) medium supplemented with 100 μg/mL Ampicillin at 37°C while shaking at 200 rpm. The culture was inoculated with a 1:100 dilution of preculture and grown overnight for 16 h to a final OD600 of 5.5 to 6.5. Fresh cell culture was used for clarification with the AcroPrep™ CCSF plates.
Like the yeast, we lysed E. coli cells from part of the culture before adding to the filter plate by pelleting them using centrifugation at 1000 × g for 5 to 10 min. Following this, we slowly decanted the supernatant to be replaced with an identical volume of lysing buffer, consisting of Lysozyme at 2 mg/mL in TE buffer composed of 30 mM Tris-HCl and 1 mM EDTA, pH 7.5 to 8.5. This mixture was then incubated in a ThermoMixer C instrument for 30 min at 25°C under shaking.
Clarification of cell cultures and filtration of lysates with AcroPrep™ 24-well CCSF plates
We added fresh (intact) cell cultures from S. cerevisiae or E. coli, as well as their lysates, into the AcroPrep™ CCSF plate (7 mL/well for vacuum, 6 mL/well for centrifuge) and processed them by vacuum (15 inHg) on a multiwell plate manifold, or by centrifugation at 1000 × g for up to 7 min. Table 1 describes the number of samples that we tested.
We analyzed both the initial cultures, lysates, and the filtrates collected in the receiver plates, for several parameters to assess the effectiveness of the clarification/filtration process with the AcroPrep™ CCSF plate.
Table 1. Sample distribution over AcroPrep™ CCSF plates and wells/plate analyzed for studies determining filtration efficiency of fresh intact cultures and lysed cells of S. cerevisiae and E. coli, with processing taking place by vacuum (Vac) or centrifugation (Cent)
| Action | Property | S. cerevisiae | E. coli | ||||||
| Intact | Lysed | Intact | Lysed | ||||||
| Vac | Cent | Vac | Cent | Vac | Cent | Vac | Cent | ||
| Sample loaded | Plates | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
| Wells/plate | 22 | 22 | 2 | 2 | 24 | 24 | 24 | 24 | |
| Wells/plate analyzed | Processing time | 22 | 22 | 2 | 2 | 24 | 24 | 24 | 24 |
| Hold-up volume | |||||||||
| Protein recovery | 3 | 3 | 2 | 2 | 3 | 3 | 3 | 3 | |
| Turbidity | |||||||||
| pH | |||||||||
| Conductivity | |||||||||
Quality parameters
- Processing time: We monitored the processing time by adding cell culture to each well and filtering it using a vacuum (15 inHg). We recorded the processing time for the first and last wells to complete filtration and reported the data as the average processing time. For centrifugation, we conducted an initial 5 min spin at 1000 × g, increasing in further increments of 2 min until no visible liquid remained in the 24-well filter plate.
- pH/Conductivity: We measured pH and conductivity of cell culture before and after filtration using a commercially available pH/conductivity meter.
- Optical density: We took OD600 readings of cultures and lysates before and after processing by vacuum or by centrifugation.
- Turbidimetry: We measured turbidity of cell cultures and lysates before and after filtration using a turbidimeter. We pooled samples from two wells together to achieve the volume necessary for measurement (10 mL).
- Total protein recovery: We determined total protein recovery by measuring the protein concentration before and after filtration using a fluorometer and protein assay kit. For raw cell cultures, we could not accurately determine the initial protein concentration (upstream), likely due to interference by cell debris present in the media. To alleviate this, we increased the protein concentration of the initial cell cultures by adding bovine γ-globulin at a concentration of 0.3mg/mL as determined by fluorometry. We then removed cells and debris from a small aliquot by filtration through a 0.2 µm syringe filter to facilitate the measurement of the initial protein concentration and allow the calculation of protein recovery.
Hold-up volume
We estimated the hold-up volume (Eq 1) - the unrecoverable sample remaining after filtration, by weighing the plate, assuming a culture density of 1 g/mL. Direct filter plate weights were unreliable, as some trapped liquid leaks out when the outlet tips contact a surface.
Calculation using plate weights:
A. Net weight filter plate and collection plate: Collection plate + filter plate (empty/no solution)
B. Net weight collection plate: Collection plate (empty/no solution)
C. Gross weight filter plate, collection plate, and sample: Collection plate + filter plate (after filtration)
D. Gross weight receiver plate + filtrate: Collection plate (after filtration)
E. Total volume: Collection plate + filter plate (after filtration) (C) - Collection plate + filter plate (empty/no solution) (A)
F. Filtrate volume: Collection plate (after filtration) (D) - Collection plate (empty/no solution) (B)
Hold-up volume = Total volume (E) – Filtrate volume (F)Eq 1. Calculation of hold-up volume
Results and discussion
Table 2 shows the results of our experiments with S. cerevisiae for cell clearance from raw cultures (intact), and filtration of lysed cells. For intact cells, each data point is the average ± standard deviation (SD) of 12 wells from four plates whereas for lysed cells each data point is the average ± SD of 8 wells from four plates. Table 3 shows the results of similar experiments with E. coli TOP10/pUC19. Each data point is the average ± SD of data generated of 12 wells from four plates.
For both organisms, filtering with the AcroPrep™ CCSF plates led to a significant reduction in turbidity and OD600, reflecting effective intact cell removal from raw culture and debris removal from the lysed cell preparations. Moreover, we observed only minimal changes in conductivity or pH, indicating that filtration with the AcroPrep™ CCSF plates did not cause significant cell lysis or allow salts to leach from the filter material.
Additionally, for both S. cerevisiae and E. coli, we noticed that filtration of cell lysates in AcroPrep™ CCSF plates took longer than the clearance of intact cells. This was true for both processing by centrifugation and by vacuum. The increased filtration time for lysates versus intact cells was most notable for the vacuum processing of S. cerevisiae, where processing the lysate took approximately four times longer than clearing intact cells. Interestingly, we noted that the total protein concentration of the initial input and filtrate did not differ significantly.
We found that protein recovery for both S. cerevisiae and E. coli samples, after filtration of lysates processed by vacuum, was greater than 88% and better than 95% for the clearance of intact cells processed by vacuum or centrifugation. The hold-up volume during centrifugation was consistently larger than that observed under vacuum.
This difference might be due to the varied orientation of the plates during filtration, as plates in the swinging bucket rotor are oriented almost sideways at a different angle than when filtering on the vacuum manifold.
Table 2. Filtration of raw culture (intact cells) and lysates (lysed cells) of S. cerevisiae through AcroPrep™ 24-well CCSF plates
| Property (unit) | Intact cells | Lysed cells | ||||
|
Initial
|
Centrifuge (1000 × g) |
Vacuum (15 inHg) |
Initial
|
Centrifuge (1000 × g) |
Vacuum (15 inHg) |
|
| Hold-up volume (µL) | N.A | 450 ± 18 | 329 ± 29 | N.A. | N.D. | N.D. |
| Processing time (min) | N.A. | 5 | < 4 | N.A. | 7 | < 16 |
| Conductivity (µS/min) | 3641 ± 30 | 3725 ± 12 | 3721 ± 15 | 5663 ± 10 | 5693 ± 14 | 5804 ± 35 |
| pH | 5.9 ± 0.0 | 5.8 ± 0.1 | 5.0 ± 0.0 | 5.3 ± 0.0 | 5.5 ± 0.0 | 5.7 ± 0.0 |
| OD600 (AU) | 33.7 ± 0.5 | 0.0 ± 0.0 | 0.1 ± 0.1 | 33.6 ± 0.8 | 0.0 ± 0.0 | 0.0 ± 0.0 |
| Turbidity (NTU) | 3903 ± 71 | 1.4 ± 0.1 | 1.5 ± 0.2 | 589 ± 113 | 11.6 ± 0.4 | 6.5 ± 1.1 |
| Total protein conc. (mg/mL) | 0.45 ± 0.0 | 0.45 ± 0.0 | 0.44 ± 0.0 | 1.11 ± 0.1 | 1.01 ± 0.0 | 0.98 ± 0.0 |
| Total protein recovery (%) | N.A. | 99.9 ± 1.5 | 98.0 ± 1.4 | N.A. | 91.5 ± 2.2 | 88.4 ± 3.0 |
Table 3. Filtration through AcroPrep™ 24-well CCSF plates of raw intact culture and lysates of E. coli TOP10/pUC19
| Property (unit) | Intact cells | Lysed cells | ||||
|
Initial
|
Centrifuge (1000 × g) |
Vacuum (15 inHg) |
Initial
|
Centrifuge (1000 × g) |
Vacuum (15 inHg) |
|
| Hold-up volume (µL) | N.A | 385 ± 44 | 229 ± 15 | N.A. | 391 ± 10 | 230 ± 90 |
| Processing time (min) | N.A. | 5 | < 8 | N.A. | 5 | < 11 |
| Conductivity (µS/min) | 17 614 ± 25 | 18 589 ± 170 | 18 298 ± 255 | 2021 ± 11 | 2029 ± 9 | 2040 ± 20 |
| pH | 6.9 ± 0.0 | 6.9 ± 0.0 | 6.9 ± 0.0 | 8.4 ± 0.0 | 8.3 ± 0.0 | 8.3 ± 0.0 |
| OD600 (AU) | 6.1 ± 0.5 | 0.1 ± 0.0 | 0.1 ± 0.0 | 5.8 ± 0.2 | 0.0 ± 0.0 | 0.0 ± 0.0 |
| Turbidity (NTU) | 1700 ± 48.5 | 2.3 ± 0.6 | 2.7 ± 1.8 | 913 ± 19 | 1.5 ± 0.2 | 1.0 ± 0.1 |
| Total protein conc. (mg/mL) | 0.40 ± 0.0 | 0.38 ± 0.0 | 0.38 ± 0.0 | 0.43 ± 0.0 | 0.42 ± 0.0 | 0.38 ± 0.0 |
| Total protein recovery (%) | N.A. | 96.8 ± 1.5 | 95.2 ± 1.3 | N.A. | 98.3 ± 0.2 | 89.2 ± 1.8 |
Conclusions
In this application note, we aimed to assess the efficiency of the AcroPrep™ CCSF plates to clarify cell cultures and filter lysates of S. cerevisiae and E. coli. Processing took place under vacuum and by centrifugation.
We conclude that AcroPrep™ CCSF plates were efficient at clarifying both lysed and intact cell cultures of S. cerevisiae and E. coli, as indicated by reductions of turbidity and OD600, with similar results for both processing methods and a total protein recovery of more than 88% for all groups. Processing time varied mainly according to the cell treatment with longer time for lysed cells cultures. Hold up volume was less for the plates used under vacuum than in centrifugation.
Our AcroPrep™ CCSF plates proved very effective for clearance of cell cultures and filtration of lysates of both yeast and bacteria further broadening their application range.
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