Toward biotherapeutic product real-time quality monitoring

Biotherapeutics, such as those derived from monoclonal antibodies (mAbs), are industrially produced in controlled multiunit operation bioprocesses. Each unit operation contributes to the final characteristics of the bioproduct. The complexity of the bioprocesses, the cellular machinery, and the bioproduct molecules, typically leads to inherent heterogeneity and variability of the final critical quality attributes (CQAs). In order to improve process control and increase product quality assurance, online and real-time monitoring of product CQAs is most relevant. In this review, the recent advances in CQAs monitoring of biotherapeutic drugs, with emphasis on mAbs, and throughout, the different bioprocess unit operations are reviewed. Recent analytical techniques used for assessment of product-related CQAs of mAbs are considered in light of the analytical speed and ability to measure different CQAs. Furthermore, the state of art modeling approaches for CQA estimation in real-time are presented as a viable alternative for real-time bioproduct CQA monitoring under the process analytical technology and quality-by-design frameworks in the biopharmaceutical industry, which have recently been demonstrated.     

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and -ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.     

Unit Operation Optimization for the Manufacturing of Botanical Injections Using a Design Space Approach: A Case Study of Water Precipitation

Quality by design (QbD) concept is a paradigm for the improvement of botanical injection quality control. In this work, water precipitation process for the manufacturing of Xueshuantong injection, a botanical injection made from Notoginseng Radix et Rhizoma, was optimized using a design space approach as a sample. Saponin recovery and total saponin purity (TSP) in supernatant were identified as the critical quality attributes (CQAs) of water precipitation using a risk assessment for all the processes of Xueshuantong injection. An Ishikawa diagram and experiments of fractional factorial design were applied to determine critical process parameters (CPPs). Dry matter content of concentrated extract (DMCC), amount of water added (AWA), and stirring speed (SS) were identified as CPPs. Box-Behnken designed experiments were carried out to develop models between CPPs and process CQAs. Determination coefficients were higher than 0.86 for all the models. High TSP in supernatant can be obtained when DMCC is low and SS is high. Saponin recoveries decreased as DMCC increased. Incomplete collection of supernatant was the main reason for the loss of saponins. Design space was calculated using a Monte-Carlo simulation method with acceptable probability of 0.90. Recommended normal operation region are located in DMCC of 0.38–0.41 g/g, AWA of 3.7–4.9 g/g, and SS of 280–350 rpm, with a probability more than 0.919 to attain CQA criteria. Verification experiment results showed that operating DMCC, SS, and AWA within design space can attain CQA criteria with high probability.     

Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature

The identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.     

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.     

New partial sequences of phosphoenolpyruvate carboxylase as molecular phylogenetic markers

To better understand the evolution of the enzyme phosphoenolpyruvate carboxylase (PEPC) and to test its versatility as a molecular character in phylogenetic and taxonomic studies, we have characterized and compared 70 new partial PEPC nucleotide and amino acid sequences (about 1100 bp of the 3' side of the gene) from 50 plant species (24 species of Bryophyta, 1 of Pteridophyta, and 25 of Spermatophyta). Together with previously published data, the new set of sequences allowed us to construct the up to now most complete phylogenetic tree of PEPC, where the PEPC sequences cluster according to both the taxonomic positions of the donor plants and the assumed specific function of the PEPC isoforms. Altogether, the study further strengthens the view that PEPC sequences can provide interesting information for the reconstruction of phylogenetic relations between organisms and metabolic pathways. To avoid confusion in future discussion, we propose a new nomenclature for the denotation of PEPC isoforms.     

Optimization of the Ethanol Recycling Reflux Extraction Process for Saponins Using a Design Space Approach

A solvent recycling reflux extraction process for Panax notoginseng was optimized using a design space approach to improve the batch-to-batch consistency of the extract. Saponin yields, total saponin purity, and pigment yield were defined as the process critical quality attributes (CQAs). Ethanol content, extraction time, and the ratio of the recycling ethanol flow rate and initial solvent volume in the extraction tank (RES) were identified as the critical process parameters (CPPs) via quantitative risk assessment. Box-Behnken design experiments were performed. Quadratic models between CPPs and process CQAs were developed, with determination coefficients higher than 0.88. As the ethanol concentration decreases, saponin yields first increase and then decrease. A longer extraction time leads to higher yields of the ginsenosides Rb₁ and Rd. The total saponin purity increases as the ethanol concentration increases. The pigment yield increases as the ethanol concentration decreases or extraction time increases. The design space was calculated using a Monte-Carlo simulation method with an acceptable probability of 0.90. Normal operation ranges to attain process CQA criteria with a probability of more than 0.914 are recommended as follows: ethanol content of 79–82%, extraction time of 6.1–7.1 h, and RES of 0.039–0.040 min⁻¹. Most of the results of the verification experiments agreed well with the predictions. The verification experiment results showed that the selection of proper operating ethanol content, extraction time, and RES within the design space can ensure that the CQA criteria are met.     

Implementation of functional genomics for gene discovery in alkaloid producing plants

Two centuries after the discovery of the first alkaloids, many enzymes involved in plant alkaloid biosynthesis have been identified. Nevertheless, the biosynthetic pathways for most of the plant alkaloids still remain incompletely characterised and understanding the regulatory mechanisms controlling the onset and flux of alkaloid biosynthesis is virtually inexistent. This information is however crucial to allow modelling of metabolic networks and predictive metabolic engineering. In the postgenomics era, new functional genomics tools, enabling comprehensive investigations of biological systems, are continuously emerging and are now gradually being implemented in the field of plant secondary metabolism as well. Here we discuss the advances these promising new technologies have already brought and may still bring with regard to the dissection of plant alkaloid biosynthesis. Encouraging results were obtained in alkaloid producing species such as Papaver somniferum, Catharanthus roseus and Nicotiana tabacum. Therefore we anticipate that functional genomics and the knowledge it brings along, will eventually allow a better exploitation of the plant biosynthetic machinery.     

Structure Elucidation of Two New Norlignans from Anemone vitifolia and Their Anti‐Inflammatory Activities

Two new norlignans together with two known phenylpropanoids were isolated from the whole herb of Anemone vitifolia. All compounds were reported from this plant for the first time. The structures of these compounds were identified by comprehensive HR‐ESI‐MS, 1D and 2D NMR spectroscopic data analysis and comparison with literature data. Additionally, bioactivity study results showed that two new compounds have potential anti‐inflammatory activity. The plausible biosynthetic pathway for these compounds were also speculated in this article.     

Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach

Quality by Design (QbD) is a new approach to the development of recombinant therapeutic protein products that promotes a better understanding of the product and its manufacturing process. The first step in the QbD approach consists in identifying the critical quality attributes (CQA), i.e., those quality attributes of the product that have an impact on its clinical efficacy or safety. CQAs are identified through a science-based risk assessment taking into consideration a combination of clinical and nonclinical data obtained with the molecule or other similar molecules or platform products, as well as the published literature. The purpose of this article is to perform a comprehensive review of the published literature, supporting an assessment of the impact on safety and efficacy of the quality attributes commonly encountered in recombinant therapeutic proteins, more specifically those produced in mammalian cell expression systems. Quality attributes generally observed in biopharmaceutical proteins including product-related impurities and substances, process-related impurities, product attributes, and contaminants are evaluated one by one for their impact on biological activity, pharmacokinetics and pharmacodynamics, immunogenicity, and overall safety/toxicity.     

Native peptide mapping – A simple method to routinely monitor higher order structure changes and relation to functional activity

ABSTRACT

In the biopharmaceutical environment, controlling the Critical Quality Attributes (CQA) of a product is essential to prevent changes that affect its safety or efficacy. Physico-chemical techniques and bioassays are used to screen and monitor these CQAs. The higher order structure (HOS) is a CQA that is typically studied using techniques that are not commonly considered amenable to quality control laboratories. Here, we propose a peptide mapping-based method, named native peptide mapping, which could be considered as straightforward for HOS analysis and applicable for IgG4 and IgG1 antibodies. The method was demonstrated to be fit-for-purpose as a stability-indicating assay by showing differences at the peptide level between stressed and unstressed material. The unfolding pathway induced by a heat stress was also studied via native peptide mapping assay. Furthermore, we demonstrated the structure–activity relationship between HOS and biological activity by analyzing different types of stressed samples with a cell-based assay and the native peptide mapping. The correlation between both sets of results was highlighted by monitoring peptides located in the complementary-determining regions and the relative potency of the biotherapeutic product. This relationship represents a useful approach to interrogate the criticality of HOS as a CQA of a drug.     

Antioxidant properties of prunes (Prunus domestica L.) and their constituents

Prunes contain large amounts of phenolics and show high antioxidant activity. The aim of this study is to clarify the contents of caffeoylquinic acid (CQA) isomers, and to estimate the contribution of these isomers to the antioxidant activity of prunes. Furthermore, structural elucidation and evaluation of antioxidant activity of prune components were also performed. CQA isomers in prunes were quantified by HPLC analysis, and it has become apparent that prunes contain relatively high amount of 4-O-caffeoylquinic acid. The contribution of CQA isomers to the antioxidant activity of prunes was revealed to be 28.4% on the basis of oxygen radical absorbance capacity (ORAC); hence, it was indicated that residual ORAC is dependent on unknown antioxidant components. Total 28 compounds were isolated and their structures were elucidated by NMR and MS analyses. Four abscisic acid related compounds, a chromanon, and a bipyrrole were novel. Each CQA isomer in prunes showed high antioxidant activities when measured by the oil stability index (OSI) method, O2- scavenging activity, and ORAC. Other isolated compounds such as hydroxycinnamic acids, benzoic acids, coumarins, lignans, and flavonoid showed high ORAC values. Furthermore, a novel chromanon indicated a remarkable synergistic effect on ORAC of CQA isomers.     

Protective effects of caffeoylquinic acids on the aggregation and neurotoxicity of the 42-residue amyloid β-protein

Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of 42-mer amyloid β-protein (Aβ42). Aβ42 aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Aβ42 oligomer, an intermediate of the aggregates, causes memory loss and synaptotoxicity in AD. Inhibition of Aβ42 aggregation by small molecules is thus a promising strategy for the treatment of AD. Caffeoylquinic acid (CQA), a phenylpropanoid found widely in natural sources including foods, shows various biological activities such as anti-oxidative ability. Previously, our group reported that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA) rescued the cognitive impairment in senescence-accelerated-prone mice 8. However, structure–activity relationship of CQA derivatives on the aggregation and neurotoxicity of Aβ42 remains elusive. To evaluate the anti-amyloidogenic property of CQA-related compounds for AD therapy, we examined the effect of CQA and its derivatives on the aggregation and neurotoxicity of Aβ42. In particular, 4,5-di-O-caffeoylquinic acid (4,5-di-CQA) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA) strongly inhibited the aggregation of Aβ42 in a dose-dependent manner. Structure–activity relationship studies suggested that the caffeoyl group in CQA is essential for the inhibitory activity. These CQAs also suppressed the transformation into β-sheet and cytotoxicity against human neuroblastoma cells of Aβ42. Furthermore, 3,4,5-tri-CQA blocked the formation of Aβ42 oligomer. These results indicate that 3,4,5-tri-CQA could be a potential agent for the prevention of AD.     

Delineation of gilvocarcin, jadomycin, and landomycin pathways through combinatorial biosynthetic enzymology

The exact sequence of events in biosyntheses of natural products is essential not only to understand and learn from nature's strategies and tricks to assemble complex natural products, but also for yield optimization of desired natural products, and for pathway engineering and muta-synthetic preparation of analogues of bioactive natural products. Biosyntheses of natural products were classically studied applying in vivo experiments, usually by combining incorporation experiments with stable-isotope labeled precursors with cross-feeding experiments of putative intermediates. Later genetic studies were dominant, which consist of gene cluster determination and analysis of gene inactivation experiments. From such studies various biosynthetic pathways were proposed, to a large extent just through in silico analyses of the biosynthetic gene clusters after DNA sequencing. Investigations of the complex biosyntheses of the angucycline group anticancer drugs landomycin, jadomycin and gilvocarcin revealed that in vivo and in silico studies were insufficient to delineate the true biosynthetic sequence of events. Neither was it possible to unambiguously assign enzyme activities, especially where multiple functional enzymes were involved. However, many of the intriguing ambiguities could be solved after in vitro reconstitution of major segments of these pathways, and subsequent systematic variations of the used enzyme mixtures. This method has been recently termed 'combinatorial biosynthetic enzymology'.     

Deoxysugar pathway interchange for erythromycin analogues heterologously produced through Escherichia coli

The overall erythromycin biosynthetic pathway can be sub-divided into macrocyclic polyketide formation and polyketide tailoring to produce the final bioactive molecule. In this study, the native deoxysugar tailoring reactions were exchanged for the purpose of demonstrating the production of alternative final erythromycin compounds. Both the d-desosamine and l-mycarose deoxysugar pathways were replaced with the alternative d-mycaminose and d-olivose pathways to produce new erythromycin analogues through the Escherichia coli heterologous system. Both analogues exhibited bioactivity against multiple antibiotic-resistant Bacillus subtilis strains. Besides demonstrating an intrinsic flexibility for the biosynthetic system to accommodate alternative tailoring pathways, the results offer an initial attempt to leverage the E. coli platform for erythromycin analogue production.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.     

Instant coffee extract with high chlorogenic acids content inhibits hepatic G‐6‐Pase in vitro,but does not reduce the glycaemia

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5‐caffeoylquinic acid (5‐CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose‐6‐phosphatase (G‐6‐Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G‐6‐Pase activity and liver glucose output induced by 5‐CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G‐6‐Pase activity of the hepatocyte microsomal fraction in a dose‐dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5‐CQA (1 mM) reduced (p < 0.05) the activity of microsomal G‐6‐Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G‐6‐Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G‐6‐Pase and reduce the liver glucose output. Copyright © 2015 John Wiley & Sons, Ltd.     

Biosynthesis of cutin monomers: involvement of a lipoxygenase/peroxygenase pathway

Cutin is synthesized from oxygenated fatty acids derived preponderantly from oleic acid. The enzymatic pathways involved in the biosynthesis of such cutin monomers have been studied, i.e. 18-hydroxyoleic acid, 9,10-epoxy-18-hydroxystearic acid (the major constituent) and 9,10,18-trihydroxystearic acid. This was approached by studying (i) the substrate specificity and stereoselectivity of purified peroxygenase, which epoxidizes unsaturated fatty acids, and fatty acid epoxide hydrolase, i.e. two enzyme activities that have been found recently in higher plants, and (ii) the transformation of oleic acid into cutin monomers by a cell free system, i.e. soybean microsomes. These two enzymes, along with a ω-hydroxylating activity, can account for the biosynthesis of the oleic acid-derived cutin monomers and their precursors. A new biosynthetic scheme is proposed, whose pathways take into account the dynamic aspects of the expression of the different enzyme activities involved. Importantly, since peroxygenase, for its activity, is strictly dependent on fatty acid hydroperoxides, which act as co-substrates, the biosynthesis of cutin monomers is also dependent on the activity of lipoxygenases.     

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance

During the process of terpene biosynthesis, C–C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the ‘biosynthetic history’ of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ¹³C value for FDP, based on δ¹³C values and relative amounts of the products, was identical with its measured δ¹³C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon–carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without ‘touching’ the plant.     

Characterization of TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase from the D-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies

Deoxysugars are critical structural elements for the bioactivity of many natural products. Ongoing work on elucidating a variety of deoxysugar biosynthetic pathways has paved the way for manipulation of these pathways for the generation of structurally diverse glycosylated natural products. In the course of this work, the biosynthesis of d-mycaminose in the tylosin pathway of Streptomyces fradiae was investigated. Attempts to reconstitute the entire mycaminose biosynthetic machinery in a heterologous host led to the discovery of a previously overlooked gene, tyl1a, encoding an enzyme thought to convert TDP-4-keto-6-deoxy-d-glucose to TDP-3-keto-6-deoxy-d-glucose, a 3,4-ketoisomerization reaction in the pathway. Tyl1a has now been overexpressed, purified, and assayed, and its activity has been verified by product analysis. Incubation of Tyl1a and the C-3 aminotransferase TylB, the next enzyme in the pathway, produced TDP-3-amino-3,6-dideoxy-d-glucose, confirming that these two enzymes act sequentially. Steady state kinetic parameters of the Tyl1a-catalyzed reaction were determined, and the ability of Tyl1a and TylB to process a C-2 deoxygenated substrate and a CDP-linked substrate was also demonstrated. Enzymes catalyzing 3,4-ketoisomerization of hexoses represent a new class of enzymes involved in unusual sugar biosynthesis. The fact that Tyl1a exhibits a relaxed substrate specificity holds potential for future deoxysugar biosynthetic engineering endeavors.