Endotoxins are the major contributors to the pyrogenic response caused by contaminated pharmaceutical products, formulation ingredients, and medical devices. Recombinant biopharmaceutical products are manufactured using living organisms, including Gram‐negative bacteria. Upon the death of a Gram‐negative bacterium, endotoxins (also known as lipopolysaccharides) in the outer cell membrane are released into the lysate where they can interact with and form bonds with biomolecules, including target therapeutic compounds. Endotoxin contamination of biologic products may also occur through water, raw materials such as excipients, media, additives, sera, equipment, containers closure systems, and expression systems used in manufacturing. The manufacturing process is, therefore, in critical need of methods to reduce and remove endotoxins by monitoring raw materials and in‐process intermediates at critical steps, in addition to final drug product release testing. This review paper highlights a discussion on three major topics about endotoxin detection techniques, upstream processes for the production of therapeutic molecules, and downstream processes to eliminate endotoxins during product purification. Finally, we have evaluated the effectiveness of endotoxin removal processes from a perspective of high purity and low cost.

The presence of endotoxin, also known as lipopolysaccharides (LPS), as a side product appears to be a major drawback for the production of certain biomolecules that are essential for research, pharmaceutical, and industrial applications. In the biotechnology industry, gram-negative bacteria ( e . g ., Escherichia coli ) are widely used to produce recombinant products such as proteins, plasmid DNAs and vaccines. These products are contaminated with LPS, which may cause side effects when administered to animals or humans. Purification of LPS often suffers from product loss. For this reason, special attention must be paid when purifying proteins aiming a product as free as possible of LPS with high product recovery. Although there are a number of methods for removing LPS, the question about how LPS removal can be carried out in an efficient and economical way is still one of the most intriguing issues and has no satisfactory solution yet. In this work, polymeric poly-ε-caprolactone (PCL) nanoparticles (NPs) ( d P = 780 ± 285 nm ) were synthesized at a relatively low cost and demonstrated to possess sufficient binding sites for LPS adsorption and removal with ~100% protein recovery. The PCL NPs removed greater than 90% LPS from protein solutions suspended in water using only one milligram (mg) of NPs, which was equivalent to ~1.5 × 10 6 endotoxin units (EU) per mg of particle. The LPS removal efficacy increased to a higher level (~100%) when phosphate buffered saline (PBS containing 137 mM NaCl) was used as a protein suspending medium in place of water, reflecting positive effects of increasing ionic strength on LPS binding interactions and adsorption. The results further showed that the PCL NPs not only achieved 100% LPS removal but also ~100% protein recovery for a wide concentration range from 20–1000 μg/ml of protein solutions. The NPs were highly effective in different buffers and pHs. To scale up the process further, PCL NPs were incorporated into a supporting cellulose membrane which promoted LPS adsorption further up to ~100% just by running the LPS-containing water through the membrane under gravity. Its adsorption capacity was 2.8 × 10 6 mg of PCL NPs, approximately 2 -fold higher than that of NPs alone. This is the first demonstration of endotoxin separation with high protein recovery using polymer NPs and the NP-based portable filters, which provide strong adsorptive interactions for LPS removal from protein solutions. Additional features of these NPs and membranes are biocompatible (environment friendly) recyclable after repeated elution and adsorption with no significant changes in LPS removal efficiencies. The results indicate that PCL NPs are an effective LPS adsorbent in powder and membrane forms, which have great potential to be employed in large-scale applications.

The focus of this work is to develop a technology for the synthesis of polymer microcarriers that demonstrate mammalian cell culture adhesion on the surface of the microcarriers. Most mammalian cells are adherent in nature that requires multilayer vessels, large volume, expensive cell culture media, high manufacturing time, and high costs of cell culture supplies for the commercial-scale manufacturing of cells. The development of an efficient, scalable technology for producing large volumes of cells is a need in bioprocess industries to improve product potency. We developed a method of synthesizing soft biocompatible US FDA approved polymer based microparticle carrier system of approximately 260 ± 27 μm in diameter that serves as an adherent platform for human umbilical vein endothelial cells (HUVEC) to grow in suspension. Our preliminary experimental results showed that using the polymeric microcarrier system cell adhesion to the surface of the microcarriers was 2−3-fold higher than conventional cell culture flasks while using 10-fold lower cell culture media in a bioreactor than a tissue-culture treated flask. The survival of HUVEC on microparticles was confirmed by live cell staining (green fluorescent calcein AM), dead cell staining (ethidium homodimer-1), nuclear DAPI staining, actin cytoskeleton staining, confocal microscopy, and flow cytometry analysis. This technology will provide high cell culture productivity while reducing the costs of growing adherent cells.

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The goal of this work is to synthesize a multifunctional biofilter and assess its efficiency to remove endotoxins, freshwater harmful algal bloom (FHAB)-derived cyanotoxins, and toxic heavy metal ions from water using polymeric nanoparticle (PolyBall) embedded in a soy protein isolate (SPI)-derived cellulose acetate (CA) biofilter. The presence of even a trace number of endotoxins in biopharmaceutical solutions can create a pyrogenic response in the human body by triggering the immune system’s signaling cascade. The purification of products usually involves sequential ion-exchange chromatography, gel filtration, and/or ultrafiltration that are complex and expensive and are associated with high (>40%) product loss. The water treatment plants use a series of standard cleansing procedures to diminish cyanotoxins; however, finding an effective and affordable technique remains a challenge. We have developed a PolyBall scavenger of endotoxins, based on nonporous poly-ε-caprolactone (PCL) polymer nanoparticles (PCL NPs) that are capable of removing endotoxins from protein solutions at high efficiency (>99%) while maintaining >99% protein recovery. In this work, we put our efforts incorporating PCL NPs (PolyBalls) in SPI and CA biofilters to achieve improved efficiency in removing endotoxins, cyanotoxins, and toxic heavy metal ions such as lead ions from contaminated solutions. Six different recombinant protein solutions of molecular weights varying from 14 to 341 kDa and with isoelectric points (pI) varying from 4.5 to 10.7 were spiked with 5.74 × 105 EU/mL of endotoxins. The contaminated protein solutions were passed through biofilters that showed >99% of endotoxin removal from the solutions while maintaining >99% protein recovery. The biofilter exhibited excellent capability in cyanotoxin separation from surface water collected from three different lake water. This is the first demonstration of soy protein plant-based portable filters with embedded biocompatible polymeric NPs, which provide strong adsorptive interactions for toxin removal from protein solutions and surface water.