Ion exchange chromatography
Anion exchange chromatography can be used to separate negatively charged endotoxin molecules from positively charged molecules, such as basic proteins. Proteins exhibit different charges at different pHs. A protein exhibits a neutral charge if the pH is equal to its isoelectric point (pI), a negative charge if the pH is > its pI, and a positive charge if the pH is < its pI 116. The pI of an endotoxin molecule is ~2 16,20,117, meaning endotoxins are negatively charged under conditions typically encountered during chromatography. At pH < 2, the target protein exhibits a net positive charge and is repelled by a positive stationary phase while the negatively charged endotoxins interact with the stationary phase and leaves the column at a lower velocity 118,119. Anion exchange chromatography is not ideal for removing endotoxins from negatively charged target molecules, such as pDNA or acidic proteins 120,121.
If significant ionic interactions are present between target proteins and endotoxins or between the protein and the resin, a decrease in protein yield or an insufficient separation may be observed. If the protein and the endotoxin have a strong interaction, endotoxins leave the column bound to the target protein. If there is a strong attractive interaction between the target protein and the resin, the protein yield is low 117.
To lessen undesirable interactions, the pH of the protein solution is adjusted. The effects of resin volume and contact time in addition to pH and conductivity on the efficiencies of endotoxin removal have been explored for therapeutic products like, antigens NY-ESO-1, Melan-A, and SSX-2 117. The pIs of these antigens were 9.1, 8.7, and 6.2, respectively. NY-ESO-1 and Melan-A are both hydrophobic molecules while SSX-2 is hydrophilic 117. All tests were run using equilibrated Q XL resin. An increase in resin volume and endotoxin-resin contact time had a positive effect on endotoxin removal and the concentration of endotoxins in the permeate consistently decreased with increase in above variables. Low endotoxin concentration of ~ 0.4 EU/µg was obtained in the permeate and a protein recovery of > 80 % was obtained consistently at almost all resin volumes 117. While positively charged proteins are less likely to interact with the resin and remain in the column, they may also demonstrate an undesirable attraction to endotoxins. To minimize protein- endotoxin interactions, the pH chosen should be high enough to avoid giving the protein a strong positive charge. Effect of different pHs on the removal of endotoxin from protein Melan-A, a hydrophobic protein with a pI of 8.7 has been studied. Melan-A exhibited a strong ionic interaction with endotoxins below its pI, causing endotoxins to leave the column with the target protein. To remedy this, the pH was increased to weaken such interactions 117. The pH tested were 7.9, 8.4, 8.9, and 9.2, which corresponded to endotoxin concentrations in the permeate of 1.4, 1.8, 0.6, and 0.5 EU/µg 117. As the pH was increased above the protein’s pI, the endotoxin concentration decreased dramatically and with no significant impact on the protein yield 117.
The success of ion-exchange chromatography is highly dependent on the target molecule, but in general ion-exchange chromatography can achieve an endotoxin reduction of five orders of magnitude for concentrated solutions (>10,00 EU/ml) or three to four orders of magnitude from dilute endotoxin solutions (<100 EU/ml) 20.
Affinity Chromatography
Affinity chromatography is used to separate endotoxins from target molecules using highly specific interactions between endotoxins and a ligand bound to a stationary phase 122. Because of the specificity of the ligand, there is little to no product loss during separation 35. The target therapeutic molecule will elute with a greater velocity than endotoxin molecules due to specificity. The ligand chosen should have a strong interaction with endotoxins and a weak interaction with the target therapeutic molecule at separation conditions. Affinity chromatography is applicable to a wide range of target molecules, including proteins and pDNA 123,124.
It is important to note that the exact structure of endotoxins varies between bacteria strains based on the core polysaccharides and the long chain polysaccharide. For this reason, ligands are typically designed to interact 20 with the most conserved section 23,25,29,125 of the endotoxin molecule, Lipid A , through hydrophobic 117 and electrostatic interactions 20. Common ligands used in affinity chromatography include Polymyxin-B, histidine, dimethylamine ligands, deoxycholic acid and polycationic ligands 17,126. Hydrophobic polymers in the form of nanoparticles have been explored for removing endotoxins from water and protein solutions.100,101
One of the most commonly used ligands is Polymyxin-B (PMB), a cyclic lipopeptide with a high affinity for endotoxin (Figure 6 ). As a ligand, PMB induces the dissociation of endotoxin aggregates 127 and binds to the Lipid A section of endotoxins 128 through hydrophobic interactions 129. PMB’s affinity to endotoxin can be attributed to the terminal amidine groups that are spaced such that interactions between amidine groups and the two phosphate groups on Lipid A can occur simultaneously 130. In addition to being used as a ligand, PMB is an antibiotic used to treat gram-negative bacterial infections. Despite PMB’s high affinity for endotoxin, columns utilizing PMB may experience a higher than average product loss 20. This is because there are positive charges on the amino acid groups on PMB that may attract negatively charged target molecules. Additionally, PMB is both neurotoxic and nephrotoxic, which may cause a problem if the ligand is released from the column 25. Work has been going on to develop peptides with similar compositions to PMB but with a decreased toxicity. These peptide analogs displayed a strong affinity to endotoxin as well as a decreased lethality when introduced intravenously into mice 131.
The nitrogenous bases adenine, cytosine, histidine and histamine all display an affinity for endotoxin. Of these, histamine and histidine are equally as effective as polymyxin B and have been successful with separating endotoxin molecules from albumin, insulin, lysozyme, myoglobin, and others. Although histamine and histidine are considered equally effective, histamine is biologically active so histidine may be preferred 20. Deoxycholic acid (DOC) is another ligand option that may offer a higher product recovery due to a low charge density that reduces ionic interactions with negatively charged proteins 20.
Cost should be considered when choosing a ligand. If the ligand cost is too high, it will not be profitable on a manufacturing scale. Additionally, the contact time required between the solution and ligand will affect the cost. A process with a high contact time will required a larger column and therefore a greater initial investment.
The pore size of the resin should also be considered. A small pore size will increase the retention of endotoxin in the column by size exclusion, while larger pore sizes will reduce the ionic interactions with anionic proteins 20. Studies have been conducted to study the effect of pH and ionic strength solutions on endotoxin removal efficiencies from hemoglobin samples using an Acticlean Etox affinity column. Endotoxins have been reported to form stable complexes with hemoglobin, thus complicating separation 43,132.The effect of ionic strength on endotoxin removal efficiency and hemoglobin recovery have been studied using two different salt solutions (NaCl and CaCl2). The endotoxin removal efficiency displayed a decreasing trend as the ionic strength was increased. However, the endotoxin removal efficiency for CaCl2 solutions displayed a more drastic initial decrease than that for NaCl solutions. These results indicate that not only do ionic interactions play a role in affinity chromatography, but the types of cations matter as well 43.
Unlike the endotoxin removal efficiency, the ionic strength and cation type had a limited effect on the product recovery from hemoglobin-endotoxin solutions. For all endotoxin contaminated hemoglobin solutions tested, the recovery of hemoglobin showed an increasing trend as the ionic strength was increased. Beyond, the ionic strength of 0.10 M, the hemoglobin recoveries remained relatively constant or displayed a gradual decrease with values over 95%. Though there existed interactions between endotoxins and hemoglobin that hindered separation but all the endotoxin contaminated hemoglobin solutions prepared with either NaCl or CaCl2 had hemoglobin recoveries above 99% for ionic strengths of 0.1 M, indicating that there is both an attraction between hemoglobin and the affinity resin and between hemoglobin and endotoxin which are weakened at an ionic strength of 0.1 M 43.
The effect of pH on endotoxin removal efficiency and hemoglobin recovery was tested using different buffer solutions. The endotoxin removal efficiency of resins was governed by the pI. There was a continuous and gradual decrease in endotoxin removal efficiency as the pH was increased from 4.5 to 8, and then the removal efficiency plummeted when the pH was increased from 8 to 9 because the pI of the affinity resin was 8. As the pH was increased from 4.5 to 8, the resin became less positively charged and was therefore less effective at attracting negatively charged endotoxins through electrostatic interactions but other affinity mechanisms were still present. As the pH was increased beyond 8, the resin moved from having a neutral charge to a negative charge that repelled endotoxins and overpowered some of the attractive affinity interactions. On the other hand, the pH or pI had a minimal effect on hemoglobin recovery; the recovery of hemoglobin from endotoxin solutions was above 97% for all pHs tested 43,130.
Commercial resins employing hydrophobic and/or cationic ligands to remove endotoxin from proteins and biological solutions use porous nano and/or microparticles and have shown great promise in protein purification, but the type of ligand immobilized or incorporated within the matrix still governs its intravenous application. Many of these resins have shown reasonable endotoxin binding efficiency from therapeutic proteins and biological solutions but suffer from major shortcomings like low recombinant protein recovery and difficulty in intravenous application due to nephrotoxicity and neurotoxicity associated with the endotoxin binding ligands. Toxicity related shortcoming can surely be addressed by using biocompatible endotoxin selective polymers which are non-toxic. Another major concern associated with most of the porous resins used for endotoxin removal is that they come in packed bed form which suffer from major drawbacks like high pressure drop (due to combined effect of bed consolidation and column blinding) and poor mass transfer (as intraparticle diffusion is responsible for transport of solute to the binding sites), thus making their application expensive and adding significant cost to downstream purification.
The toxicity, pressure drop and mass transfer related shortcomings were addressed by using biocompatible, rigid and non-porous particles where adsorption takes place on the surface. One such study focused on using biocompatible and environment friendly polymer, poly-ε-caprolactone (PCL) nanoparticles ~ 800 nm to remove endotoxins from water and protein solutions 100,101. The PCL nanoparticles (PolyBalls) were non-porous in nature and thus the endotoxin binding took place on the surface of the particles (Figure 7 (a) ). PolyBalls showed high endotoxin removal efficiency of >99% from phosphate buffer saline (PBS) solution. These particles were also effective in removing endotoxin from protein solution prepared in water with more than 90% removal efficiency 100. The removal efficiency was >99% when protein solutions were prepared in phosphate buffer saline (PBS) 100. The research also reported high endotoxin binding capacity of ~\(1.5\times 10^{6}\) endotoxin unit (EU) per mg of particles 100. In addition to high endotoxin removal the particles offered high protein recovery in excess of 90% thus maximizing therapeutic product recovery. High endotoxin removal in presence of PBS was attributed to the creation of shielding effect in presence of lyotropic sodium chloride salt. Considering larger-scale industry applications, combinatorial techniques were applied to construct PolyBall containing flexible and multifunctional biofilters (Figures 7 and 8 ). Contaminated samples were allowed to flow from one side of the filter to the other. The kinetics of endotoxin removal efficiency were determined as a function of concentration that also removed >99% endotoxins from water. One major advantage of the biocompatible PolyBalls and multifunctional biofilters is that they can be reused for endotoxin binding quite effectively without a major loss in binding efficiency. PolyBalls can be regenerated by breaking endotoxin-nanoparticle complexes which makes the endotoxin removal process more efficient and scalable. Figure 8(e) andTable 1 showcase a comparison of different endotoxin removal products in terms of binding capacity, protein recovery and cost. Although, non-porous particles solve the mass transfer related limitation but the problem of high pressure drop during purification operation still persists. Due to the specificity of the ligand, affinity interactions offer a low product loss with a wide range of applications. Both mixed-mode chromatography and membrane adsorption use similar mechanisms and experience benefits.
Mixed-Mode Chromatography
Mixed-mode chromatography is a growing separation technique in the biopharmaceutical industry 45,133,134. While traditional chromatographic methods rely on a single dominant interaction between the ligand and the targeted molecule, mixed-mode chromatography (MMC) utilizes multiple interaction modes for an increased separation 45,134,135. When compared to traditional chromatographic methods, MMC offers an increased retention and selectivity of the targeted compound 136,137, especially for polar charged molecules 133,134. Many ligands used in affinity chromatography, such as histamine and histidine can be considered mixed-mode ligands due to their beneficial secondary interactions 121,137-139.