Biologic formulations are scrupulously examined because the drug product is injected into the blood stream. This mode of drug delivery evades a natural defence mechanism of the body to ingest any foreign entity. Hence it is axiomatic to have a stricter microbial limit tests and elemental impurities than oral dosages. Additionally, an imperative parameter that needs to be tested is endotoxins. These are lipopolysaccharides present in the cell wall of bacteria which illicit a primary biological response to induce specific systems. They trigger an immune response from the body and manifest a range of pathological features. Endotoxins in the final drug product can be present through two sources. They can originate from the process related impurities or from excipients which are added in the final formulations. This review summarises the following parameters of endotoxins: structure, mechanism of action, sources, testing methods, unit operations for removal of endotoxins and the need of utilising low endotoxin excipients.
Review
Bacteria are a class of unicellular organisms which are present everywhere in nature. There are many types of bacteria, they can either help our body in digesting fiber present in food, lactose present in milk and they can also cause infection in our body. Bacteria are predominantly classified as gram negative and gram-positive bacteria. Gram negative bacteria can cause infection in our body with the help of endotoxins. Endotoxins are a class of macromolecules called Lipopolysaccharides (LPS). LPS are present in the outer membrane of gram-negative bacteria A typical E.Coli cell possesses ∼2 × 106 LPS molecules, covering about three quarters of the cell membrane. It has been estimated that 70,000 molecules/min are exported to the outer membrane to sustain the growth rate of an organism. These are important macromolecules with a substantial number of cellular resources dedicated to their production. Endotoxins are released in the environment when bacterial cell is disrupted or in other words when cell lysis occurs.
Structurally LPS consists of three structural domains: lipid A, the core oligosaccharide and O antigen. Lipid A is the hydrophobic portion of the molecule, it is bounded to the lipid outer membrane. It is released from the dividing cells and from the lysed bacterial cells. Lipid A elicits inflammatory responses in human body upon recognition by the complex Toll-like receptor 4 and myeloid differentiation factor (TLR4-MD2). This factor is predominantly found on macrophages, monocytes and dendritic cells. Lipid A is linked to core oligosaccharide by Beta-1-6 linkage to glucosamine disaccharide. Core oligosaccharide forms the extra cellular region of the outer membrane.
The core polysaccharide has a non-repeating oligosaccharide that is linked to glucosamines of Lipid A. The oligosaccharide structure usually contains Kdo residues, heptoses and various hexoses which can be modified with phosphates and other substituents such as phosphoethanolamine. The core oligosaccharide chains are important to bacteria for transporting LPS to the outer membrane and resistance to antibiotics.
The final part of LPS is O antigen and it contains a repeating oligosaccharide of two to eight sugars. The lipid A structure is conserved, core polysaccharide vary among species and O antigen is the most diverse component of LPS. The structure and composition of antigen differ within a species at strain level. O antigen contributes to bypassing the host immune defences.
LPS elicit primary biological response to induce specific symptoms and pathologies of the disease. It brings about a potent and pleiotropic stimulus of host immune system. The immune responses depend on the cell type to which LPS is bound. In monocytes and macrophages following responses are triggered:
- Production of inflammatory cytokines like prostaglandins and leukotrienes lead to inflammation.
- Complement activation initiates histamine release causing vasodilation.
- Activation of blood coagulation cascade which cause inflammation, haemorrhage, intravascular coagulation and septic shock.
Endotoxins are measured by Limulus Amoebocyte Lysate (LAL) Test. LAL is an extract from blood cells of horseshoe crab. These crabs are returned to their natural habitat after extraction of LAL. According to FDA, US and Europe pharmacopeia, parenteral formulations have to be tested for bacterial endotoxins by LAL test. Endotoxins limits for parenteral applications are defined in the monographs. LAL test for oral dosage forms is not mandatory as endotoxins are degraded in the stomach.
Drug delivery in biopharmaceuticals is always through intravenous, intramuscular and intradermal. Hence the formulation will be tested for endotoxins according to the pharmacopeia. When the product is biologically produced, the incomplete removal of microorganisms during purification can result in drug substance having high endotoxin levels. Therefore, the need of identifying the sources of endotoxins and their removal during purification is paramount. Usually, the sources of endotoxins are the water used as the solvent or in the processing, packaging components, excipients, buffers, cell culture medium, reagents, serum, supplements. A common source of endotoxin contamination is the water system that grows biofilms bacteria on the walls of water distribution pipelines. Endotoxins are highly stable and resistant to degradation by heat or pH. They form stable interaction with target therapeutic compound that further complicates separations. Endotoxins forms aggregates in micelle, cube, lamellar or vesicle forms exhibiting a negative charge in the solution. These micellar endotoxins can be adsorbed on polycationic ligands on various surfaces and on therapeutic protein as well. Hence endotoxin removal is a challenging task as they form stable interactions with themselves and with target therapeutics. Following are the modes by which endotoxins are purified in biopharmaceutical downstream processing.
Ultrafiltration
Depending on the size of core polysaccharide, a single endotoxin moiety can have a molecular weight between 10-30 kDa. The micellar and vesicle structure can form an aggregate upwards of 1000 kDa and diameters up to 0.1 um. These high molecular weight structures can be separated by ultrafiltration. Ultrafiltration can be used to separate endotoxins from small therapeutic drug molecules like BMS‐753493 (Epothilone–Folic Acid Conjugate). During this process smaller endotoxin molecules filter through the membrane while the bigger aggregates of endotoxins are present in the retentate. Efficiency of endotoxin removal increases with dilution of protein solution. This happens due to the shift in equilibrium from endotoxin aggregates intro monomers in dilute solutions. The shortcoming with ultrafiltration is its limited endotoxin removal efficiency for molecules that are much smaller than endotoxin aggregates. This method is suited for removing endotoxins from small molecule therapeutics which do not have affinity for endotoxins.
Extraction
This is used to separate endotoxins from target therapeutics based on their relative solubilities in two immiscible liquids. Endotoxins are extracted in the organic phase and the target molecule remains in the aqueous phase. Isothermal solvent extraction is effective is removing endotoxins in purifying plasmids. Solvent extraction technique is efficient in removing endotoxins but the product yield in protein solutions is significantly low. Low yield in protein solutions can be attributed to denaturation of protein in repeated heating and cooling cycles as well as change in protein conformation if a surfactant is used.
Ion Exchange chromatography
Endotoxins exhibit a net negative charge because of its Antigen moiety. Proteins have a different charge at different pH due to its amino acids group present on the exterior surface. The protein will exhibit a positive charge if the pH is set lower than its pI (Isoelectric point). Hence anion exchange chromatography can be used to separate negatively charged endotoxins from positively charged proteins. The net positive charge of the protein will not interact with the stationary phase and leave the column during the wash phase of chromatography. Decrease in the product yield or inefficient separation is observed if there is a strong interaction between endotoxins and protein or if the protein is acidic in nature. In both cases change in the pH to make protein net positive can remedy the situation. In general, ion exchange chromatography is efficient in removing endotoxins. It is reported to reduce the concentration of endotoxins in 3 to 5 orders of magnitude depending upon the dilution of protein solution.
Affinity chromatography
This is used to separate endotoxins from the product of interest using specific interactions between ligand bound in stationary phase and endotoxins. This specificity ensures minimal to no product loss during carrying out this procedure. Hence it is imperative to have ligand chosen has a strong affinity to endotoxin and a weak affinity towards the product. The structure of endotoxin varies based on core polysaccharide and the antigen. Therefore, ligands are designed to interact with the most conserved structure on a endotoxin molecule. The ligands have an affinity towards the Lipid A through hydrophobic and electrostatic interactions. Commonly used ligands in affinity chromatography include Polymyxin b, dimethylamine ligands, histidine, deoxycholic acid and hydrophobic polymer nanoparticles. Porous nanoparticles and microparticles are used in commercial resins employing hydrophobic and cationic ligands to remove endotoxins. Few of the ligands have shown to have a nephrotoxicity and neurotoxicity in intravenous applications. Drawbacks of using a porous resin include a high pressure drop and poor mass transfer of adsorbate in adsorbent pores. These problems can be solved by using a biocompatible, rigid and non-porous particles where interaction happens on the surface.
Membrane adsorption
This adopts the principles of chemistry used in affinity and ion-exchange chromatography but offers a reduced processing time. The support membrane has same ligands used in membrane separation are the same as affinity chromatography or resins used in the ion exchange chromatography. The product yields are similar to those achieved in affinity chromatography. Processing times are made faster compared to chromatography by improving flow rates and eliminating diffusion limitations. Membranes are made of cellulose, cellulose acetate, polyvinyl alcohol, Polyvinyl acetate and Polyvinylidene fluoride (PVDF). There is no need of cleaning or regenerating the membrane as theses membranes are single-use. Hence increased flow rates and reduced processing steps contribute to streamlining the operation to make it faster than chromatography.
Removal and detection of endotoxins from a biopharmaceutical manufacturing process is an intensive procedure. Hence purity of excipients for product stabilisation added in membrane separation and final formulation becomes imperative. Formulators prefer to have more stringent limits on endotoxins than mentioned in pharmacopeia. Nuances in packaging materials are also considered to further mitigate risk.
Signet have made strategic partnerships by keeping the industry requirements in consideration to provide high purity low endotoxin excipients and raw materials. It has partnered with renowned manufacturers like Pfanstiehl, Roquette, Meggle, Galactic and Novo Nordisk. These manufacturers ensure multi-compendial, high purity endotoxins with lot to lot consistency and produced by complying with ICH Q7 guidelines. The portfolio is designed in such a way that the products can be utilised from inoculum development to final formulation. The application wise product range is as follows-
Upstream process:
Downstream and Final formulation: