Removing endotoxins using a spin-column format

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Thermo Fisher Scientific, Rockford, IL

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Rizwan Farooqui

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Removing endotoxins using a spin-column format

Fast and effective removal of endotoxins from protein samples.

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

July 9, 2012


Endotoxin removal during the development of biological therapeutics is critical for preventing inflammation, tissue death and endotoxin shock. Traditional resins that use polymixin-B to remove endotoxins have low binding efficiency. Using gravity-flow chromatography can improve binding by increasing the contact time of the resin with the endotoxin-containing sample. As with most biological samples, however, minimizing processing time is important for preventing sample degradation via proteolysis, denaturation or aggregation.

This study’s goal was to develop a relatively fast method for removing endotoxins from samples that results in high protein recovery. Although poly(ε-lysine) has affinity for lipopolysaccharides (endotoxins), a streamlined procedure for its use to treat biological samples was lacking. The Thermo Scientific Pierce Endotoxin Removal Resin is an affinity resin that consists of spherical, rigid beads with immobilized poly(ε-lysine) (Figure 1). The resin is composed of environmentally friendly natural polymer cellulose and an FDA-approved food preservative, poly(ε-lysine). To evaluate the resin, we tested sample incubation time, resin-to-sample ratio, maximum endotoxin load, and the ability to regenerate the resin. We also compared the resin to other commercially available endotoxin removal methods.

Schematic of the poly(ε-lysine) affinity ligand immobilized onto a porous cellulose resinFigure 1. Schematic of the poly(ε-lysine) affinity ligand immobilized onto a porous cellulose resin. Endotoxins bind via ionic and hydrophobic interactions. The multiple ε-aminobutyl groups impart both a positive charge via the primary amines and a hydrophobic characteristic via the butyl spacer arm. The hydrophilic characteristic of the cellulose base-matrix is partially masked by thorough derivatization of its interior and exterior surfaces with the poly(ε-lysine).


RESULTS and DISCUSSION:

FDA guidelines dictate that the endotoxin unit (EU) level in therapeutic material must be ≤5EU/mL per kilogram of body weight. To determine the incubation time required to reduce endotoxin levels from 10,000EU/mL to ≤5EU/mL, we measured the levels at different incubation periods using bovine serum albumin (BSA) samples spiked with 10,000EU/mL. At 30 minutes incubation with the resin, endotoxin levels decreased below 5EU/mL and, thereafter, endotoxin levels decreased marginally (Figure 2). Furthermore, to determine the limits of the 1-hour spin protocol, we tested BSA samples with endotoxin levels from 5000 to 500,000EU/mL. At all the levels, removal of >98% of endotoxins resulted; however, final endotoxin units per milliliter was above FDA guidelines when the initial concentration was ≥50,000 (Table 1). Protein recovery was >90%.

Figure 2. Endotoxins are effectively removed after a 30-minute incubation.

Figure 2. Endotoxins are effectively removed after a 30-minute incubation. The Pierce High Capacity Endotoxin Removal Spin Columns (0.5mL) were incubated with BSA (1mL at 1mg/mL) spiked with 10,000EU. One endotoxin unit (EU) equals approximately 0.1ng of endotoxin.

Table 1. Endotoxins are effectively removed from highly contaminated samples. BSA (2mL of 1mg/mL) samples were spiked with endotoxins and processed with the Pierce High Capacity Endotoxin Removal Spin Columns (0.5mL).  
Inital Endotoxin Concentration (EU/mL) Final Endotoxin Concentration (EU/mL) Endotoxin Removal Efficiency (%)
5000 < 1 99.98
12,500 < 1 99.99
25,000 1.26 99.99
50,000 7.1 99.99
250,000 32 99.98
500,000 9600 98.08

 

To determine the optimal resin-to-sample ratio for efficient endotoxin removal, we added various volumes of BSA containing 10,000EU/mL to the Pierce High Capacity Endotoxin Removal Resin. When sample volumes were equal to the resin volume (1:1), final endotoxin levels after 1 hour incubation were greater than 10EU/mL. To effectively reduce endotoxin levels to >5EU/mL, a minimum of 1:2 resin-to-sample ratio was required (Table 2).

 

Table 2. The resin-to-sample ratio affects endotoxin removal effectiveness. Different volumes of BSA at 1mg/mL containing 10,000EU/mL were processed with spin columns containing different volumes of Pierce High Capacity Endotoxin Removal Resin.
Resin Volume (mL) Sample Volume (mL) Resin-to-sample Ratio Final Endotoxin Concentration (EU/mL)
1 1 1:1 > 10
1 2 1:2 < 5
1 4 1:4 < 1
1 8 1:8 < 1
0.5 0.5 1:1 > 10
0.5 1 1:2 < 5
0.5 4 1:8 < 1
0.25 0.25 1:1 >10
0.25 0.5 1:2 < 5
0.25 1 1:4 < 1

 

We also evaluated resins from other suppliers. The Pierce High Capacity Endotoxin Removal Resin was more effective for removing endotoxins than the other resins tested. Furthermore, most of the resins from other suppliers resulted in more protein loss (Table 3).

 

Table 3. The Thermo Scientific Pierce High Capacity Endotoxin Removal Resin is more effective than resins from other suppliers. BSA samples (2mL of 1mg/mL) containing 25,000 EU/mL were processed with the indicated resins according to the manufacturers’ instructions.
Resin/Source Final Endotoxin Concentration (EU/mL) Protein Recovery (%)
Pierce High Capacity Endotoxin Removal Resin < 1 91
Thermo Scientific Detoxi-Gel Enotoxin Removal Gel 77 74
Affi-Prep* Polymyxin Matrix (Bio-Rad) 13 88
Polymyxin B-Agarose (Sigma) 900 63
ReductEtox* (Sterogene) 13,900 92
EndoTrap* red (Hyglos) 64 82

 

Endotoxin removal resins are often re-used and require a clean-up step to eliminate the risk of contaminating other samples. To monitor the resin’s performance after multiple regenerations, we performed five cycles of endotoxin removal and regenerated the resin via a 2-hour or 12-hour (overnight) protocol. The Pierce High Capacity Endotoxin Removal Resin can be regenerated with 0.2M NaOH in 95% ethanol for two hours or with 0.2M NaOH in endotoxin-free water for 12 hours. In all tests, endotoxin removal was >99% and protein recovery was >85% (Table 4).

 

Table 4. The endotoxin removal resin maintains effectiveness after multiple regenerations. Samples contained BSA (1mL at 1mg/mL) spiked with 10,000EU. The final endotoxin concentration for each cycle and method was <1EU/mL).
Regeneration Cycle Protein Recovery (%) 12 hour Protocol Protein Recovery (%) 2 hour Protocol
1 96 88
2 91 86
3 89 89
4 88 90
5 89 88

CONCLUSIONS:

The Pierce High Capacity Endotoxin Removal Resin effectively reduces endotoxin levels in samples and performs better than other commercially available methods. The spin-column format makes the removal process fast, enabling effective removal from BSA samples using a 30-minute incubation; however, the standard protocol uses a 1-hour incubation, which might be necessary for some sample types. The resin is stable and can be regenerated at least five times without loss in performance. Therefore, if a sample’s endotoxin level is greater than the acceptable limits after processing, the resin can be regenerated and the sample processed again.


METHODS:

Endotoxin removal: The spin column containing the resin was centrifuged at 500 x g for 1 minute to remove the storage buffer. The column was washed with either 0.2N NaOH (overnight) or 0.2N NaOH in 95% ethanol for 1-2 hours and then with 2M NaCl followed by endotoxin-free water. The resin was equilibrated three times with endotoxin-free phosphate-buffered saline. The sample was added and incubated at 4-22°C with gentle end-over-end mixing for 30 minutes to 1 hour. The column was centrifuged at 500 x g for 1 minute to collect the sample. Resins from other suppliers were used according to the manufacturers’ instructions. Endotoxin levels were measured using the Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit (Part No. 88282).

Sample-to-resin ratio: The spin-column pre-packed with different volumes of Pierce High Capacity Endotoxin Removal Resin was challenged with different volumes of BSA at 1mg/mL containing 10,000EU/mL in phosphate-buffered saline (50mM sodium phosphate, 0.15M sodium chloride, 0.05% sodium azide; pH 7.2). Samples were incubated for 1 hour with constant mixing. The protein solution was recovered by centrifugation. Endotoxin levels were measured using the Pierce LAL Chromogenic Endotoxin Quantitation Kit.

Resin regeneration: The Pierce High Capacity Endotoxin Removal Resin was used for multiple cycles of endotoxin removal and subsequent resin regeneration with 0.2M NaOH for 12 hours or 0.2M NaOH in 95% ethanol for 2 hours. Samples contained 1mL of BSA (1mg/mL) spiked with 10,000EU of E. coli strain 055:B5 in phosphate-buffered saline (10mM sodium phosphate, 0.15M sodium chloride, 0.05% sodium azide; pH 7.2). This cycle was repeated five times with each regeneration treatment. Endotoxin levels were measured using the Pierce LAL Chromogenic Endotoxin Quantitation Kit, and protein concentration was determined using the Pierce BCA Protein Assay (Part No. 23225).

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