Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

Amino acid supplementation for enhancing recombinant protein production in E. coli.

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent-like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth-promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 µL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

High-throughput screening of active compounds against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 ±â€¯1.41 and 3.13 ±â€¯0.98 µM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

First demonstration of the circulation of a pneumovirus in French pigs by detection of anti-swine orthopneumovirus nucleoprotein antibodies.

The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

A fluorometric method for the assay of protein kinase activity.

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.

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.

Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli.

The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

Use of a fluorescent probe to monitor the enhanced affinity of rh-BMP-2 to silicated-calcium phosphate synthetic bone graft substitutes under competitive conditions.

A comparative investigation was undertaken on 1-2mm sized granules of two forms of synthetic bone graft substitute (SBG) with identical pore structure but varied bulk chemistry, stoichiometric hydroxyapatite (HA) and silicate substituted (0.8wt% Si) hydroxyapatite (SA), to assess the influence of SBG chemistry on the relative affinity of an osteogenic growth factor (GF), recombinant human bone morphogenetic protein-2 (rhBMP-2). A previously described novel fluorescent probe, fluoresceinthioureidoaminocaproic acid (FTCA), was covalently attached to rhBMP-2 to give FTCA-rhBMP-2 and facilitate the quantitative monitoring of GF uptake and release from the two chemistries of SBG. The relative affinity of rhBMP-2 for the HA and SA granules was assessed at a physiologically relevant concentration of 300ngmL-1 from three (increasingly complex) environments; phosphate buffered saline (PBS), minimum Eagles' medium (MEM) and serum supplemented MEM (SCEM) in order to closely mimic clinical bone repair procedures. The results demonstrated that rhBMP-2 affinity to SBGs was highly sensitive to both SBG chemistry and the composition of the local environment. Under the most physiologically relevant competitive conditions of SCEM, rhBMP-2 showed greater affinity to SA (P<0.05) such that 50% of the rhBMP-2 in solution was adsorbed to the SA granules after only 15min, as compared to 30% adsorbed to the HA granules. Subsequent investigation of the desorption of adsorbed GF from the SBGs demonstrated that a significantly higher percentage of the adsorbed rhBMP-2 was desorbed from HA as compared to SA granules. Together, these observations suggested that at physiologically relevant concentrations and conditions, rhBMP-2 has a greater affinity to silicate-substituted hydroxyapatite as compared to stoichiometric hydroxyapatite, which may in part explain the enhanced osteoconductivity and reported osteoinductivity for silicate-substituted hydroxyapatite based SBGs.

Colonization study of gfp-tagged Achromobacter marplatensis strain in sugar beet.

This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA3, MeJA and Ag+, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Biosimilars for psoriasis: preclinical analytical assessment to determine similarity.

Biosimilars, sometimes called 'generic biologics', are no longer a vision for the future but a present-day reality. Drug manufacturers and regulatory authorities are charged with ensuring that these products are safe and effective. Because biologically produced medications are large, complex proteins, many factors affect the quality of the end product, including glycosylation and presence of impurities, and thus many factors need to be compared between an emerging biosimilar and its originator biologic. Indeed, preclinical analytical assessments to determine similarity to an originator biologic are critical and are considered to be the foundation for regulatory approval of biosimilars. Here, the science behind the preclinical development of biosimilars is discussed by members of the International Psoriasis Council, and suggestions are put forth to try to ensure that future biosimilars are produced in a high quality and standardized manner.

Subvisible (2-100 µm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Nanowired Delivery of Growth Hormone Attenuates Pathophysiology of Spinal Cord Injury and Enhances Insulin-Like Growth Factor-1 Concentration in the Plasma and the Spinal Cord.

Previous studies from our laboratory showed that topical application of growth hormone (GH) induced neuroprotection 5 h after spinal cord injury (SCI) in a rat model. Since nanodelivery of drugs exerts superior neuroprotective effects, a possibility exists that nanodelivery of GH will induce long-term neuroprotection after a focal SCI. SCI induces GH deficiency that is coupled with insulin-like growth factor-1 (IGF-1) reduction in the plasma. Thus, an exogenous supplement of GH in SCI may enhance the IGF-1 levels in the cord and induce neuroprotection. In the present investigation, we delivered TiO2-nanowired growth hormone (NWGH) after a longitudinal incision of the right dorsal horn at the T10-11 segments in anesthetized rats and compared the results with normal GH therapy on IGF-1 and GH contents in the plasma and in the cord in relation to blood-spinal cord barrier (BSCB) disruption, edema formation, and neuronal injuries. Our results showed a progressive decline in IGF-1 and GH contents in the plasma and the T9 and T12 segments of the cord 12 and 24 h after SCI. Marked increase in the BSCB breakdown, as revealed by extravasation of Evans blue and radioiodine, was seen at these time points after SCI in association with edema and neuronal injuries. Administration of NWGH markedly enhanced the IGF-1 levels and GH contents in plasma and cord after SCI, whereas normal GH was unable to enhance IGF-1 or GH levels 12 or 24 h after SCI. Interestingly, NWGH was also able to reduce BSCB disruption, edema formation, and neuronal injuries after trauma. On the other hand, normal GH was ineffective on these parameters at all time points examined. Taken together, our results are the first to demonstrate that NWGH is quite effective in enhancing IGF-1 and GH levels in the cord and plasma that may be crucial in reducing pathophysiology of SCI.

Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers.

Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato.

Comparative proteomics of milk fat globule membrane proteins from transgenic cloned cattle.

The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.

Impacts of high ß-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via (13)C metabolic flux analysis.

The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high ß-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of ß-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.

Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.

Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes.