Prediction models for the flux decay profile and initial flux of microfiltration for therapeutic proteins.

Microfiltration (MF) is an essential step during biopharmaceutical manufacturing. However, unexpected flux decay can occur. Although the flux decay profile and initial flux are important factors determining MF filterability, predicting them accurately is challenging, as the root cause of unexpected flux decay remains elusive. In this study, the methodology for developing a prediction model of flux decay profiles was established. First, the filtration profiles of different monodisperse polystyrene latex and silica beads of various sizes were evaluated. These results revealed that the size and surface electrostatic properties of the beads affect the flux decay profile. Taking the size and surface electrostatic properties of protein aggregates into account, we constructed a predictive model using model bead filtration profiles. We showed that this methodology was applicable to two different MF filters to predict the flux decay profile of therapeutic proteins. Because our proposed prediction model is based on normalized flux, the initial flux is required to predict the overall filtration profile. Then, we applied the Hagen-Poiseuille equation using sample viscosity values to estimate the initial flux. The developed prediction models can be used for effective MF scale-up assessment during the early stages of process development.

Recent progress on heterologous protein production in methylotrophic yeast systems.

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha.

Scale-up analysis of geometrically dissimilar single-use bioreactors.

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.

The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

The Role of a Nonribosomal Peptide Synthetase in l-Lysine Lactamization During Capuramycin Biosynthesis.

Capuramycins are one of several known classes of natural products that contain an l-Lys-derived l-α-amino-ɛ-caprolactam (l-ACL) unit. The α-amino group of l-ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP-independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP-dependent amide-bond-forming catalyst responsible for the biosynthesis of the remaining amide bond present in l-ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l-Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS-mediated mechanism is employed in the biosynthesis of other l-ACL-containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS-derived peptides that contain an unmodified l-Lys residue.

A biocatalytic approach to capuramycin analogues by exploiting a substrate permissive N-transacylase CapW.

Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from a methyl ester precursor as an acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed that this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.

Genetic and Environmental Influences on Traits of Gender Identity Disorder: A Study of Japanese Twins Across Developmental Stages.

The present study examined: (1) gender and age differences of mean gender identity disorder (GID) trait scores in Japanese twins; (2) the validity of the prenatal hormone transfer theory, which predicts that, in dizygotic (DZ) twin pairs, twins with an opposite-gender co-twin more frequently exhibit GID traits than twins with a same-gender co-twin; and (3) the magnitude of genetic and environmental influences on GID traits as a function of age and gender. Data from 1450 male twin pairs, 1882 female twin pairs, and 1022 DZ male-female pairs ranging from 3 to 26 years of age were analyzed. To quantify individual variances in GID traits, each participant completed four questionnaire items based on criteria for GID from the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR). Our most important findings were: (1) Japanese females exhibited GID traits more frequently than males and Japanese children exhibited GID traits less frequently than adolescents and adults (among females, the prevalence was 1.6 % in children, 10 % in adolescents, and 12 % in adults; among males, the prevalence was 0.5, 2, and 3 %, respectively); (2) the data did not support the prenatal hormone transfer theory for GID traits; and (3) a large part of the variance for GID traits in children was accounted for by familial factors; however, the magnitude was found to be greater in children than in adolescents or adults, particularly among females. This study suggests that although the prevalence is likely to increase, familial effects are likely to decrease as individuals age.

Effect of pH, NaCl concentration, and mAb concentration of feed solution on the filterability of Planova™ 20N and Planova™ BioEX.

Virus filtration is one of the most important steps in ensuring viral safety during the purification of monoclonal antibodies (mAbs) and other biotherapeutics derived from mammalian cell cultures. Regarding the various virus retentive filters, including Planova filters, a great deal of data has been reported on the virus retention capability and its mechanism. Along with the virus retention capability, filterability is a key performance indicator for designing a robust and high-throughput virus filtration step. In order to obtain higher filterability, optimization of the feed solution conditions, and filter selection is essential; however, limited data are available regarding the filtration characteristics of Planova filters. Furthermore, for Planova 20N and Planova BioEX, the virus retention characteristics were reported to differ due to their respective membrane materials and layer structures. Whether these filters differ in their filtration characteristics is an interesting question, but no comparative evaluations have been reported. In this study, the filterability of the two filters was investigated and compared using 15 feed mAb solutions of a single mAb selected by design of experiments with different combinations of pH, NaCl concentration, and mAb concentration. The filterability of Planova 20N was affected not only by the feed solution viscosity, but also by the mAb aggregate content of the feed mAb solution and mAb-membrane electrostatic interactions. In contrast, the filterability of Planova BioEX decreased under some buffer conditions. These findings and the established design spaces of these filters provide valuable insights into the process optimization of virus filtration.

Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Bidirectional influences between maternal parenting and children's peer problems: a longitudinal monozygotic twin difference study.

This twin study examined the bidirectional relationship between maternal parenting behaviors and children's peer problems that were not confounded by genetic and family environmental factors. Mothers of 259 monozygotic twin pairs reported parenting behaviors and peer problems when twins were 42 and 48 months. Path analyses on monozygotic twin difference scores revealed that authoritative parenting (the presence of consistent discipline and lack of harsh parenting) and peer problems simultaneously influenced each other. Authoritative parenting reduced peer problems, and peer problems increased authoritative parenting. Neither consistent discipline nor harsh parenting alone was associated with peer problems. These results suggest that maternal authoritative parenting works protectively in regard to children's peer problems, and peer problems can evoke such effective parenting.

Two cohort and three independent anonymous twin projects at the Keio Twin Research Center (KoTReC).

The Keio Twin Research Center has conducted two longitudinal twin cohort projects and has collected three independent and anonymous twin data sets for studies of phenotypes related to psychological, socio-economic, and mental health factors. The Keio Twin Study has examined adolescent and adult cohorts, with a total of over 2,400 pairs of twins and their parents. DNA samples are available for approximately 600 of these twin pairs. The Tokyo Twin Cohort Project has followed a total of 1,600 twin pairs from infancy to early childhood. The large-scale cross-sectional twin study (CROSS) has collected data from over 4,000 twin pairs, from 3 to 26 years of age, and from two high school twin cohorts containing a total of 1,000 pairs of twins. These data sets of anonymous twin studies have mainly targeted academic performance, attitude, and social environment. The present article introduces the research designs and major findings of our center, such as genetic structures of cognitive abilities, personality traits, and academic performances, developmental effects of genes and environment on attitude, socio-cognitive ability and parenting, genes x environment interaction on attitude and conduct problem, and statistical methodological challenges and so on. We discuss the challenges in conducting twin research in Japan.

Structure-based gene targeting discovery of sphaerimicin, a bacterial translocase I inhibitor.

Rise and shine: Using a gene-targeting approach aimed at identifying potential L-threonine:uridine-5'-transaldolases that catalyze the formation of (5'S,6'S)-C-glycyluridine, a new bacterial translocase I inhibitor was discovered from an actinomycete following fermentation optimization.

Comprehensive modeling of cell culture profile using Raman spectroscopy and machine learning.

Chinese hamster ovary (CHO) cells are widely utilized in the production of antibody drugs. To ensure the production of large quantities of antibodies that meet the required specifications, it is crucial to monitor and control the levels of metabolites comprehensively during CHO cell culture. In recent years, continuous analysis methods employing on-line/in-line techniques using Raman spectroscopy have attracted attention. While these analytical methods can nondestructively monitor culture data, constructing a highly accurate measurement model for numerous components is time-consuming, making it challenging to implement in the rapid research and development of pharmaceutical manufacturing processes. In this study, we developed a comprehensive, simple, and automated method for constructing a Raman model of various components measured by LC-MS and other techniques using machine learning with Python. Preprocessing and spectral-range optimization of data for model construction (partial least square (PLS) regression) were automated and accelerated using Bayes optimization. Subsequently, models were constructed for each component using various model construction techniques, including linear regression, ridge regression, XGBoost, and neural network. This enabled the model accuracy to be improved compared with PLS regression. This automated approach allows continuous monitoring of various parameters for over 100 components, facilitating process optimization and process monitoring of CHO cells.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Characterization of LipL as a non-heme, Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that generates uridine-5'-aldehyde during A-90289 biosynthesis.

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 µM, K(m)(UMP) = 14 µM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine:uridine-5'-aldehyde transaldolase.

The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic ß-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5'-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5'-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5'-phosphate, the enzyme has no activity with alternative amino acids, such as glycine or serine, as aldol donors, and acetaldehyde is a coproduct. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5'S,6'S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

An ATP-independent strategy for amide bond formation in antibiotic biosynthesis.

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C beta-lactamase (CapW) was identified within the biosynthetic gene cluster that-in contrast to the expected beta-lactamase activity-catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine-dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond formation.

Vitamin D binding protein-macrophage activating factor inhibits HCC in SCID mice.

BACKGROUND: A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. METHODS: The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. RESULTS: DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. CONCLUSION: DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC.

Development of a stable antibody production system utilizing an Hspa5 promoter in CHO cells.

Chinese hamster ovary (CHO) cells are widely used for manufacturing antibody drugs. We attempted to clone a novel high-expression promoter for producing monoclonal antibodies (mAbs) based on transcriptome analysis to enhance the transcriptional abundance of mAb genes. The efficacy of conventional promoters such as CMV and hEF1α decrease in the latter phase of fed-batch cell culture. To overcome this, we screened genes whose expression was maintained or increased throughout the culture period. Since CHO cells have diverse genetic expression depending on the selected clone and culture medium, transcriptome analysis was performed on multiple clones and culture media anticipated to be used in mAb manufacturing. We thus acquired the Hspa5 promoter as a novel high-expression promoter, which uniquely enables mAb productivity per cell to improve late in the culture period. Productivity also improved for various IgG subclasses under Hspa5 promoter control, indicating this promoter's potential universal value for mAb production. Finally, it was suggested that mAb production with this promoter is correlated with the transcription levels of endoplasmic reticulum stress-related genes. Therefore, mAb production utilizing the Hspa5 promoter might be a new method for maintaining protein homeostasis and achieving stable expression of introduced mAb genes during fed-batch culture.

Development of a retention prediction model in ion-pair reversed-phase HPLC for nucleoside triphosphates used as mRNA vaccine raw materials.

The International Conference on Harmonization guidelines for quality on pharmaceutical development recommends a systematic development approach including robustness studies which assure performance of manufacturing and analytical method development of drug product. It was demonstrated that the retention prediction model for nucleoside triphosphates (NTPs) on ion-pair reversed-phase HPLC was developed by a highly accurate Kawabe's model which supports the development of robust HPLC methods. As NTPs and its derivatives are typically used for Messenger ribonucleic acid (mRNA) vaccine production, adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), 5-methylcytidine-5'-triphosphate (m5-CTP), uridine-5'-triphosphate (UTP), 5-methyluridine-5'-triphosphate (m5-UTP), pseudouridine-5'-triphosphate (Ψ-UTP) and N1-methylpseudouridine-5'-triphosphate (m1Ψ-UTP) were applied for prediction model development. By a comparison of the predicted retention factor in eight studied samples with the retention factor measured under six isocratic conditions, the absolute prediction error was 0.075 and also the prediction error (%) was 2.70%. In practical examples, analytical method for residual ATP, GTP, CTP, and m1Ψ-UTP in the commercial mRNA-based drugs and purity method for UTP derivatives were optimized by QbD approach. The design space for the minimum resolution between adjacent peaks was simulated with the models developed to evaluate the robustness of peak separation, and the optimal mobile phase condition was also simulated. As a conclusion, the desired peak was successfully separated under the optimized condition, and we thought that these retention models could optimize the mobile phase condition of the NTP analysis method for applying to various quality tests, such as quantity, purity and identity test for NTPs and its derivates in the mRNA-based drugs.