Carbon dioxide is often promoted as a sustainable solvent, as CO 2 is non-flammable, exhibits a relatively low toxicity and is naturally abundant. However, injudicious use of carbon dioxide in a process or product can reduce rather than enhance overall sustainability. This review specifically examines the use of CO 2 to create greener processes and products, with a focus on research and commercialization efforts performed since 1995. The literature reveals that use of CO 2 has permeated almost all facets of the chemical industry and that careful application of CO 2 technology can result in products (and processes) that are cleaner, less expensive and of higher quality.

Supercritical and near-critical CO2 in green chemical synthesis and processing

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.

https://www.tandfonline.com/doi/pdf/10.4161/mabs.2.5.12645

Recovery and purification process development for monoclonal antibody production

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(10)00031-4.pdf

Recent Advances in Large-Scale Production of Monoclonal Antibodies and Related Proteins

Future of antibody purification.

Liquid and supercritical carbon dioxide have attracted much interest as environmentally benign solvents, but their practical use has been limited by the need for high CO2 pressures to dissolve even small amounts of polar, amphiphilic, organometallic, or high-molecular-mass compounds. So-called 'CO2-philes' efficiently transport insoluble or poorly soluble materials into CO2 solvent, resulting in the development of a broad range of CO2-based processes, including homogeneous and heterogeneous polymerization, extraction of proteins and metals, and homogeneous catalysis. But as the most effective CO2-philes are expensive fluorocarbons, such as poly(perfluoroether), the commercialization of otherwise promising CO2-based processes has met with only limited success. Here we show that copolymers can act as efficient, non-fluorous CO2-philes if their constituent monomers are chosen to optimize the balance between the enthalpy and entropy of solute-copolymer and copolymer-copolymer interactions. Guided by heuristic rules regarding these interactions, we have used inexpensive propylene and CO2 to synthesize a series of poly(ether-carbonate) copolymers that readily dissolve in CO2 at low pressures. Even though non-fluorous polymers are generally assumed to be CO2-phobic, we expect that our design principles can be used to create a wide range of non-fluorous CO2-philes from low-cost raw materials, thus rendering a variety of CO2-based processes economically favourable, particularly in cases where recycling of CO2-philes is difficult.

Non-fluorous polymers with very high solubility in supercritical CO2 down to low pressures

Alternative bioseparation operations: life beyond packed-bed chromatography.

Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.

High‐throughput process development for recombinant protein purification

Carbon dioxide is a naturally abundant, environmentally benign solvent whose use, like water, in a process is not regulated by either EPA or FDA. Unfortunately, polar compounds such as amino acids and proteins are essentially insoluble in carbon dioxide. Further, alkyl-functional surfactants, which have been shown to allow extraction of proteins into conventional organic solvents, exhibit very poor or negligible solubility in CO2 at pressures below 50 MPa. Consequently, highly CO2-soluble fluoroether-functional surfactants have been generated and used to solubilize subtilisin Carlsberg from aqueous buffer and cell culture medium into CO2, with recovery accomplished by depressurization. Both the amount of protein solubilized in the emulsion and the extent of activity retention by the protein following recovery are functions of the initial protein concentration in the buffer. This, plus the observation that the presence of protein affects the stability of the emulsion, suggests that some of the protein is sacrificed to act as a stabilizer in these systems. In addition to solubilization via an inverse emulsion, it has also been shown that one can strip protein-surfactant aggregates from a middle phase emulsion using pure CO2, suggesting an ion-pairing type mechanism. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 572–580, 1998.

Landon M Steele

Papers

Alternative bioseparation operations: life beyond packed-bed chromatography.

High‐throughput process development for recombinant protein purification

Solubilization of subtilisin in CO2 using fluoroether-functional amphiphiles.