Recently, advances have been made in various areas of bioprocessing and are being used to develop efficient methods for producing recombinant proteins. These include effective bioprocess optimization using high-throughput equipment as well as disposable systems, continuous upstream processes, continuous chromatography, integrated continuous bioprocesses, quality by design and process analytical techniques to achieve higher yields of quality products. This article summarizes recent advances in recombinant protein bioprocessing, including various expression systems, bioprocess development, and upstream and downstream processes for recombinant proteins.
The downstream purification process is very important. Traditionally, recombinant proteins are purified using purification steps based on centrifugation, chromatography and membrane filtration. In addition to these steps, viral inactivation is used for recombinant biopharmaceuticals produced using mammalian cells. Cell fragmentation is necessary to recover the desired proteins expressed as intracellular inclusion bodies. A common method of cell fragmentation used on a large scale is the high-pressure homogenizer, and the purification process for recombinant proteins expressed as inclusion bodies involves the use of centrifugation to harvest the cells, cell lysis, inclusion body lysis, refolding, washer filtration, and chromatographic purification steps to obtain purified, biologically active proteins.
Centrifugation, deep filtration and tangential flow microfiltration (TFF-MF) are the most commonly used techniques for cell harvesting and cell separation. Various clarification techniques for antibody downstream processes have been described in other articles.
Refolding of proteins is necessary for the acquisition of biological activity of recombinant proteins expressed in inclusion bodies. Batch mode refolding of solubilized inclusion bodies can be performed using rapid or pulse dilution, filtration and dialysis or on-column chromatography. In a recent study, it was also determined that mild solubilization could be considered for recovery of scFv from inclusion bodies, in terms of cost, time, and label-free nature.Various methods for refolding to recover biologically active proteins have been previously reviewed. For recombinant proteins expressed extracellularly, the cell post-harvest purification steps include chromatography and filtration washing, while for recombinant proteins expressed in the periplasmic space, the steps include cell lysis, centrifugation, microfiltration, chromatographic purification, and filtration washing.
Chromatographic Processes
Various chromatographic techniques, i.e., affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography or gel filtration chromatography are used to purify recombinant protein-based biopharmaceuticals to obtain a high purity product in a biologically active form. It is well known that an increase in product concentration in the upstream process leads to an increase in the volume of the chromatographic packing and an increase in the buffer requirement.HCPs are a major source of impurities, and there are significant differences in molecular mass, charge, hydrophobicity, and structure of HCPs for each process. As a result, they pose a challenge for chromatographic purification. The yield of HCP can be reduced during upstream process development. Cell lines that produce lower levels of HCP should be selected during upstream process development to aid in efficient product purification.
Affinity chromatography (AC) is popular and is the most selective technique used to purify tagged proteins, bispecific antibodies, DNA-based biologics, cell products, viral vectors, and viruses. Some commonly used affinity tags include hexahistidine (His), glutathione S-transferase (GST), and maltose-binding protein (MBP).Protein A chromatography is the most widely used mAb purification method. The main problems associated with this chromatographic method are the shedding of the Protein A ligand and the non-specific binding of host cell proteins, DNA and other cell culture-derived impurities. Therefore, it is necessary to use other chromatographic techniques to remove these impurities. In a recent review, challenges and advances in the purification of mAb using Protein A chromatography were described, including the elevated cost of packing and its limited lifetime, Protein A ligand modifications, and alternative formats such as monolithic membranes and microspheres.
Ion exchange chromatography is the most widely used and cost-effective method for recombinant protein purification. Cation and anion exchange chromatography (CEX and AEX) removes various types of impurities such as product variants, residual HCP and DNA, media components, detached Protein A ligands, endotoxins and viruses. A study exploring the efficacy of weak anion exchangers for the separation of rHBsAg VLP from aggregated structures found rHBsAg VLP to be within acceptable quality levels of 94-97.5%. Purification of rHBsAg derived from yeast crude extracts using AEX columns resulted in high purity (up to >95%). Viral safety is a key issue for therapeutic proteins (e.g. mAb) produced using mammalian cells (e.g. CHO cells). CEX performed in overload mode has been reported to be able to remove viruses during mAb production. Hydrophobic interaction chromatography (HIC) is based on the relative hydrophobicity of protein molecules.HIC is mainly used as a purification step for recombinant protein purification. In HIC, at high ionic strengths, proteins bind to ligands, while at low ionic strengths, protein elution occurs. In a recent study, influenza A and B viruses were successfully purified by HIC using a 96-well plate format, with virus recoveries as high as 96% and residual DNA levels of approximately 1.3%. Size exclusion chromatography (SEC) or gel filtration chromatography separates protein molecules based on their molecular weight. SEC is used to purify a variety of proteins such as scFv and insulin-like growth factor receptor as well as for aggregate removal and desalting.
Membrane based chromatography process has also emerged as a good choice for recombinant protein purification. In this type of chromatography, specific ligand groups are attached to the microfiltration membrane pores. Impurities present in the protein solution bind to the membrane at a neutral to slightly basic pH and low conductivity. Optimized parameters for protein purification using membrane chromatography include membrane size distribution, thickness, and fluid flow distribution. The hydrogel and nanofiber matrices of the membranes provide high specific area, higher ligand density, and an optimized 3D binding environment. It has also been reported that the use of nanofibers in affinity chromatography membrane systems can represent a significant improvement over current viral vector purification.
It is not possible to purify proteins using single-step chromatography due to the variability of process and product-related impurities. Multi-mode or mixed-mode chromatography involves selective interactions between the chromatographic ligands and the protein molecules via ionic, hydrophobic, hydrogen bonding, or van der Waals interactions. Mixed-mode packing provides salt tolerance, better separation, and higher binding loads.Capto packing is based on hydrophobicity and ionic interactions, and ceramic hydroxyapatite (CHT) is based on electrostatic interactions and affinity interactions.MMC has been successfully used as a capture step (using hydrophobic charge chromatography) and as a purification step. Yellow fever vaccines were prepared using a bioreactor, followed by anion-exchange membrane chromatography, multimodal packing, and β-propiolactone inactivation. The overall virus recovery in these chromatography steps was 52.7%. Recently, dextran-grafted mixed-mode chromatography adsorbents have been prepared with enhanced adsorption properties for BSA/IgG.
Membrane Filtration Based Technologies
Ultrafiltration was used for purification and protein concentration, and elution filtration was used for desalting recombinant proteins. The application of a filter aid (diatomaceous earth) with cross-flow ultrafiltration to remove contaminant proteins and DNA molecules without the use of a chromatography step was also investigated. In another study, ultrafiltration/wash filtration was used for the final purification of conjugated vaccine products. A filtration-based strategy to purify influenza VLP consisting of a series of ultrafiltration and elution steps followed by a decontamination filtration step was also explored and achieved a recovery of approximately 80%. A combination of one-pass TFF concentration and AEX chromatography was also used to enhance the mAb purification step, increasing production flexibility and process productivity. An efficient chromatin-directed clarification process for cell culture fluids was developed as an alternative to Protein A chromatography for IgG purification to remove the majority of host DNA and histone proteins and to reduce non-histone HCPs, which allowed TFF to concentrate the clarified supernatant with buffer exchange, and cation-exchange chromatography to efficiently remove residual host contaminants to meet all clinical requirements.
