Analysis of Recombinant Human Serum Albumin Extraction and Degradation in Transgenic Rice Extracts.

Transgenic plant systems have successfully been used to express recombinant proteins, including rice seed-expressed recombinant human serum albumin (rHSA), without the risk of contamination of human pathogens. Developing an efficient extraction process is critical as the step determines recombinant protein concentration and purity, quantity of impurities, and process volume. This article evaluates the effect of pH and time on the extraction and stability of rHSA. The amount of rHSA in clarified extract after 60 min of solubilization increased with pH from 0.9 mg/g (pH 3.5) to 9.6 mg/g (pH 6.0), but not over time as 10 min was sufficient for solubilization. Total soluble protein in extracts also increased with pH from 3.9 mg/g (pH 3.5) to 19.7 mg/g (pH 6.0) in clarified extract. Extraction conditions that maximized rHSA purity were not optimal for rHSA stability and yield. Extraction at pH 3.5 resulted in high purity (78%), however, rHSA degraded over time. Similar purities (78%) were observed in pH 4.0 extracts yet rHSA remained stable. rHSA degradation was not observed in pH 4.5 and 6.0 extracts but higher native protein concentrations decreased purity. Strategies such as pH and temperature adjustment were effective for reducing rHSA degradation in pH 3.5 rice extracts. Low temperature pH 3.5 extraction retained high purity (97%) and rHSA stability. While seed-expressed recombinant proteins are known to be stable for up to 3 years, the degradation of rHSA was notably extensive (56% within 60 min) when extracted at low pH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:681-691, 2018.

Purification and characterization of recombinant Cel7A from maize seed.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

Recovery and purification of plant-made recombinant proteins.

Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications.

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.

Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer.

Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.

Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development.

Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)-expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin-Ciocalteu assay and by profiling on RP-HPLC. The Folin-Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP-HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA-402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts.

Factors influencing recombinant human lysozyme extraction and cation exchange adsorption.

Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5.