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. ).