Discussion
The biopharmaceuticals industry has experienced a rapid and consistent
growth over the past few years 142-145. It is
predicted that half of all drugs under development will be
biopharmaceuticals within the next 5-10 years 3.
Developing endotoxin removal methods that are both effective and cost
efficient is an ongoing challenge 45 due to the high
purity required and the potential interactions present between endotoxin
and target molecules. Affinity and mixed-mode chromatography are the
most promising methods for a widely applicable removal method due to the
highly selective interactions between endotoxins and the chosen ligand.
Additional research is still required to further develop additional
methods for removal and ligands that demonstrate a high affinity to
endotoxins with a low toxicity and cost. There is also ongoing research
to develop endotoxin-free E. coli strains that would eliminate
the need for endotoxin removal and decrease downstream processing costs
10,146,147.
Biological techniques led the way, starting with the RPT, a crude, yet
effective method of testing medicines before injecting them into humans.
This was a good start, but with a detection limit of 0.5 EU/ml, and
taking over two hours to perform, as well as requiring live rabbits for
test subjects, it was quickly outclassed by other methods. Following
close behind RPT, was LAL assay testing. This method became the industry
standard in medicine and equipment testing. It still falls short of
being fast enough to keep up with the modern world of testing needs, not
to mention the need to move away from using horseshoe crab blood in
order to protect their dwindling population. bWBA and MAT are similar to
LAL in that they fall short on keeping up with the needs of the modern
world. While they present attractive qualities, MAT being able to use
recycled monocytes from blood banks and bWBA requiring very little
preparation, they still require collection and storage of blood from
living beings. This would create difficulties in collecting proper
amounts of blood stores to handle testing requirements. Electrochemical
techniques offer nearly endless combinations of sensor and
protein-complexes, able to be designed specifically for a testing
solution that could prove difficult for other tests. Optical detection
methods offer incredibly high precision testing, with results ready in a
matter of seconds, provided the equipment can be afforded and operated
correctly. Finally, with the rise of mass-based resonance detection, the
future of detection methods rely on more accurate, real-time detection,
with increasingly cheap and easy to use.
There is no single purification method that fits all separation
scenarios 148. The method chosen will depend greatly
on the properties of the target molecule 126.
Ultrafiltration is well suited for removing endotoxins from water,
salts, or small molecule therapeutics, but it is not applicable to most
separation scenarios. Extraction provides a high endotoxin removal
efficiency for highly contaminated samples, but can possibly lead to an
undesirable level of product loss. Ion Exchange chromatography provides
adequate separation with acceptable product loss for molecules with a
weak positive charge. Due to the specificity of the ligands, affinity
chromatography and mixed-mode chromatography offer an adequate
separation with high product recovery for a wide range of target
molecules. Membrane adsorption offers a reduced processing time and
initial cost with a high product recovery, but has a low binding
capacity that limits removal efficiencies. While there is no single
method that is applicable to all scenarios, ion-exchange, affinity, and
mixed-mode chromatography all offer consistently high removal
efficiencies and product recoveries under appropriate operating
conditions. Even so, additional research is needed to develop more
widely applicable and cost-effective methods that reduce product loss
while meeting all governing regulations for endotoxin concentrations in
biopharmaceutical products.