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.