Removing endotoxins from various sample types

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Pierce High Capacity Endotoxin Removal Resin

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Thermo Fisher Scientific, Rockford, IL

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Rizwan Farooqui

Managing Editor:

Douglas Hayworth


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Eliminate endotoxins from protein and antibody samples

A highly specific affinity resin enables endotoxin removal from various protein sources, sizes and charges.

Rizwan Farooqui, Ph.D.; Babu Antharavally, Ph.D.; Alice Alegria-Schaffer, M.S.;

July 18, 2012


Traditional endotoxin removal methods are lengthy and require significant optimization for every target molecule being purified. Furthermore, ionic methods for binding endotoxins require pH optimization to avoid binding highly charged proteins, and size-exclusion methods to filter endotoxins are not effective for large proteins. We developed an endotoxin removal resin that is effective for eliminating endotoxins from protein and antibody samples. The resin is composed of environmentally friendly natural polymer cellulose and an FDA-approved food preservative, poly(ε-lysine).

We tested the Thermo Scientific Pierce High Capacity Endotoxin Removal Resin with various sample types to demonstrate its versatility. In each experiment, we performed the 1-hour spin-column method and measured the resulting endotoxin levels and protein recovery. Endotoxins were effectively removed from a variety of samples while recovering a high percentage of proteins with differing molecular weights and charges.


RESULTS and DISCUSSION:

We tested the endotoxin removal resin with four purified proteins with different molecular weights and charges. We tested each protein at a low and high endotoxin level to simulate levels in protein samples from mammalian cell and E. coli cultures, respectively. In all tests, endotoxin removal was ≥90% (Table 1) and protein recovery was ≥85% (data not shown).

Table 1. Endotoxins are effectively removed from proteins with various molecular weights and isoelectric points. Samples (1mL at 1mg/mL) were processed with the the Pierce High Capacity Endotoxin Removal Spin Columns, 0.5mL.
Protein (1mg/mL) Molecular Weight (Da) Isoelectric Point (pI) Initial Endotoxins (EU/mL)† Final Endotoxins (EU/mL) Endotoxin Removal (%)
Cytochrome C 12,000 10.6 10,000 1.35 >99
25 2.35 90
Myoglobin 17,000 6.8 10,000 3.67 >99
24 1.6 93
BSA 66,000 4.9 10,000 0.80 >99
14 0.6 96
BGG 150,000 7.4 10,000 4.60 >99
18 0.37 98
†One endotoxin unit (EU) equals approximately 0.1ng of endotoxin.

 

We also tested the resin with complex biological samples, including proteins or antibodies in cell culture supernatants, cell lysates or serum. After processing, endotoxin levels for all samples were <1EU/mL (Table 2).

 

Table 2. Effective endotoxin removal from various biological sources. Samples (2mL) were processed with the the Pierce High Capacity Endotoxin Removal Spin Columns, 0.5mL.
Protein / Source Inital Endotoxin Concentration (EU/mL) Final Endotoxin Concentration (EU/mL)
Anti-fractalkine / cell culture supernatant 8.26 <1
His-tagged GFP / E. coli 9780 <1
IgG / human serum 78 <1

 

The samples we processed had an average protein concentration of 1mg/mL, which resulted in ≥85% protein recovery after endotoxin removal. To evaluate protein recovery from dilute samples, we tested two concentrations of BSA and IgG samples (0.25mg/mL and 0.10mg/mL) with the Pierce High Capacity Endotoxin Removal Resin. We were able to remove 99% of the endotoxins; however, protein recoveries were less than the typical 85% for the 0.1mg/mL protein samples (Table 3).

 

Table 3. Protein recovery in samples with low concentrations. BSA and human IgG (2mL) containing 10,000EU of E. coli strain, 055:B5 were processed with the Pierce High Capacity Endotoxin Removal Spin Columns, 0.5mL.
Initial Protein Concentration (mg/mL) BSA Recovery (%) IgG Recovery (%)
0.25 90 89.5
0.1 72 67

 

Endotoxins are lipopolysaccharides derived from the outer membrane of gram-negative bacteria such as E. coli. Because there are numerous strains of E. coli, we tested the Pierce High Capacity Endotoxin Removal Resin for the ability to remove endotoxins from four different E. coli strains. For each strain, endotoxin removal was > 99% (Table 4).

 

Table 4. Endotoxins were effectively removed from different strains of E. coliBSA (1mL at 1mg/mL) spiked with 10,000EU from different strains of E. coli was processed with the Pierce High Capacity Endotoxin Removal Spin Columns, 0.5mL.
E. coli Strain Final Endotoxin Concentration (EU/mL) Endotoxin Removal (%) Protein Recovery (%)
011:B4 0.36 >99.9% 83%
026:B6 4.18 >99.9% 88%
0127:B8 0.87 >99.9% 87%
0128:B12 5.01 >99.9% 88%

CONCLUSIONS:

The Pierce High Capacity Endotoxin Removal Resin is effective for eliminating endotoxins from protein and antibody samples. The resin removed endotoxins from various protein sources, sizes and charges. Protein recovery was high (≥85%), even at sample concentrations as low as 0.25µg/mL. Furthermore, endotoxins from four different E. coli strains were effectively removed with this resin. The high-binding capacity and low protein retention makes this resin applicable for many protein sample types.


METHODS:

The Pierce High Capacity Endotoxin Removal Spin Columns, 0.5mL, was centrifuged at 500 x g for 1 minute to remove the storage buffer. The column was washed with 0.2N NaOH (overnight) and then with 2M NaCl followed by endotoxin-free water. The resin was equilibrated three times with endotoxin-free phosphate-buffered saline. Samples (1mL or 2mL) was added in a buffer (pH 7.2) containing 10mM sodium phosphate 0.15M sodium chloride, 0.05% sodium azide, and incubated at 22°C with gentle end-over-end mixing for 1 hour. The column was centrifuged at 500 x g for 1 minute to collect the sample. Endotoxin level was measured using the Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit (Part No. 88282) and protein recovery was determined using the Thermo Scientific Pierce BCA Protein Assay (Part No. 23225).

2 comments to Eliminate endotoxins from protein and antibody samples

  • Sophie Scohy

    Is it possible to use endotoxin removal spin column combined with chaotropic substances e.g. urea?
    Thank you in advance.

    Sophie Scohy
    Delphi Genetics
    Belgim

    • Douglas Hayworth

      That’s a good question, Sophie. We never tested this resin with samples containing urea. Because binding and removal of endotoxin with this product is based on a combination of hydrophobic and charge interaction, we suspect that urea and other chaotropes (which disrupt such interactions) would not be compatible.