Figure Legends
Figure 1: A simplified scheme of biopharmaceutical production, separation and purification steps. Biopharmaceutical manufacturing is divided into two areas: upstream fermentation or cell culture and downstream purification processes. Each area contains multiple unit operations. The primary downstream unit operation is chromatography that includes variations in modes such as affinity, cation-exchange, anion-exchange, ceramic hydroxyapatite, and hydrophobic-interaction chromatography. The process performance is mainly determined by the rate of molecule transport to the binding sites. In large chromatographic columns, small adsorbent particles provide high surface area for binding but generate a large pressure drop at high fluid velocity. On the other hand, large adsorbent particles minimize active binding site per volume as well as reduce mass transport. (Figure reproduced with permission from Jozala et. al. , Ref. 3)
Figure 2: (a) Schematic view of the chemical structure of endotoxin from E. coli . Endotoxins are lipopolysaccharides that consist of a heteropolysaccharide (O-antigen), the core oligosaccharide, and a non-polar lipid A tail. (b) Endotoxins form aggregates in micelle, cube, lamellar or vesicle forms exhibiting a net negative charge in pharmaceutical solutions. The negatively charged “micellar” endotoxins can be adsorbed on polycationic ligands, or the individual endotoxin monomers can be removed by hydrophobic lipid tail interactions with hydrophobic surface.
Figure 3 (a): Endotoxin induced defense mechanisms in circulating hemolymphs of horseshoe crabs. The LAL assay is designed based on the immunogenic reactions developed in the blood of horseshoe crabs. Upon exposure to endotoxins, the electron dense large granules (L-granule) and less electron dense small granular (S-granule) amebocytes become activated by zymogen factor C.
Figure 3 (b): Coagulation cascade in horseshoe crab blood. Endotoxin activates plasma membrane-bound factor C. Factor C is a single chain glycoprotein (M.W. = 123 kDa) comprising of a heavy chain (M.W. = 80 kDa) and light chain (M.W. = 43 kDa) that plays a major key role as an activator to immune system. Upon binding with endotoxins, an autocatalytic activity triggers with the cleavage of Phe–Ile bond resulting in an activated factor C that interacts with factor B converting it into a clotting enzyme. Clotting enzyme cleaves coagulogen at two terminal of peptide C at the Arg–Lys and Arg–Gly forming insoluble coagulin gel.
Figure 3 (c): The proteolytic activity feature of the activated clotting enzyme in horseshoe crab’s blood is used on synthetic chromogenic i.e. Gly–Arg–p-nitroaniline substrates instead of coagulogen to detect endotoxin as it separates p -Nitroaniline (p -NA). Upon addition of a chromogenic substrate, Ac-Ile-Glu-Ala-Arg-pNA, the activated protease, clotting enzyme catalyzes the release of p-nitroaniline (pNA), resulting in a yellow color that can be quantitated by measuring the absorbance at 405 nm (or absorbance at 340 nm) and extrapolating to a standard curve for correlating endotoxin concentrations.
Figure 4: (A) and (B) The design and fabrication of a new electrochemical endotoxin sensor based on a human recombinant toll-like receptor 4 (rhTLR4) and myeloid differentiation-2 (MD-2) complex. The rhTLR4/MD-2 complex, which specifically binds to endotoxin, was immobilized on gold electrodes through a self-assembled monolayer (SAM) technique involving the use of dithiobis(succinimidyl undecanoate) (DSU). (C) – (F) The electrochemical signals generated from interactions between the rhTLR4/MD-2 complex and the endotoxin were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). (G) A linear relationship between the peak current and endotoxin concentration was obtained in the range of 0.0005 to 5 EU/mL with a correlation coefficient (R 2) of 0.978. The estimated limit of detection (LOD) was fairly low, 0.0002 EU/ml. The rhTLR4/MD-2 based sensors exhibited no current responses to dipalmitoylphosphatidylcholine (DPPC) bearing two lipid chains, which is structurally similar to endotoxin, indicating the high specificity of the sensors to endotoxin. Reproduced with permission from Ref. 78.
Figure 5: Assay protocol for endotoxin detection. We have developed a fluorescence-based method that measures the changes in fluorescence intensity and the corresponding endotoxin concentration. The whole process is instantaneous and can detect endotoxin as low as 0.0001 ng/ml in solutions.
Figure 6: Distinct chemical structures are seen for the removal of endotoxins. Since endotoxins are negatively charged, anion exchange ligands are employed, e.g., diethylaminoethane (DEAE), polymyxin B, histamine, histadine, poly-l-lysine, polyethylimine (pEI) and chitosan.
Figure 7: (a) PolyBall nanoparticles are synthesized using the solvent diffusion method. (b) PolyBalls can be lyophilized in white powder form and stored at room temperature (~22). (c) PolyBalls are effective in removing >99% endotoxins (>\(2\times 10^{6}\) EU/ml) from water (dotted line) and PBS (pH 7.4) (solid line). (c) Change in LPS concentrations does not compromise PolyBall’s endotoxin removal efficiency.(d) PolyBalls efficiently remove endotoxins from a variety of protein solutions at different concentrations. (e) Removal of endotoxins does not affect protein recovery (>95% recovery) indicating minimal product loss and PolyBall’s specificity towards endotoxins even in endotoxin mixed protein solutions.(e) PolyBalls can be regenerated to remove endotoxins further. Figures reproduced with permission from Ref. 97 (Razdan et. al. ).
Figure 8: (a) PolyBall nanoparticles are embedded in a cellulose acetate (CA) biofilter. (b) Cross-sectional view of a CA filter without any nanoparticles (negative control) using SEM.(c) SEM image of a biofilter with PCL nanoparticles impregnated in it. (d) Our biofilter removes >99% endotoxins (solid line) while filter without PCL nanoparticles (negative control) is not as effective as the biofilter in removing endotoxins indicating the role of PCL nanoparticles in binding and removing endotoxins from solutions. (e) Comparison of the endotoxin removal efficiency (solid line) and protein recovery (dotted line) between our filter and other commercial endotoxin removal filters. Our filter outperforms others while removing >99% endotoxins and maintaining >95% protein recovery. Figures reproduced with permission from Ref. 97 (Razdan et. al. ).