Oral ribose supplementation in dystroglycanopathy: A single case study.

Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein (ISPD), fukutin-related protein (FKRP), and fukutin (FKTN) genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous FKRP mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation (p < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Endogenous reductase activities for the generation of ribitol-phosphate, a CDP-ribitol precursor, in mammals.

The core M3 O-mannosyl glycan on α-dystroglycan serves as the binding epitope for extracellular matrix molecules. Defects in core M3 glycans cause congenital muscular dystrophies that are collectively known as dystroglycanopathies. The core M3 glycan contains a tandem D-ribitol-5-phosphate (Rbo5P) structure, which is synthesized by the Rbo5P-transferases fukutin and fukutin-related protein using CDP-ribitol (CDP-Rbo) as a donor substrate. CDP-Rbo is synthesized from CTP and Rbo5P by CDP-Rbo pyrophosphorylase A. However, the Rbo5P biosynthesis pathway has yet to be elucidated in mammals. Here, we investigated the reductase activities toward four substrates, including ribose, ribulose, ribose-phosphate and ribulose-phosphate, to identify the intracellular Rbo5P production pathway and elucidated the role of the aldo-keto reductases AKR1A1, AKR1B1 and AKR1C1 in those pathways. It was shown that the ribose reduction pathway is the endogenous pathway that contributes most to Rbo5P production in HEK293T cells and that AKR1B1 is the major reductase in this pathway.

Improved efficacy of FKRP AAV gene therapy by combination with ribitol treatment for LGMD2I.

Mutations in the fukutin-related protein (FKRP) gene cause dystroglycanopathy, with disease severity ranging from mild LGMD2I to severe congenital muscular dystrophy. Recently, considerable progress has been made in developing experimental therapies, with adeno-associated virus (AAV) gene therapy and ribitol treatment demonstrating significant therapeutic effect. However, each treatment has its strengths and weaknesses. AAV gene therapy can achieve normal levels of transgene expression, but it requires high doses, with toxicity concerns and variable distribution. Ribitol relies on residual FKRP function and restores limited levels of matriglycan. We hypothesized that these two treatments can work synergistically to offer an optimized therapy with efficacy and safety unmatched by each treatment alone. The most effective treatment is the combination of high-dose (5e-13 vg/kg) AAV-FKRP with ribitol, whereas low dose (1e-13 vg/kg) AAV-FKRP combined with ribitol showed a 22.6% increase in positive matriglycan fibers and the greater improvement in pathology when compared to low-dose AAV-FKRP alone. Together, our results support the potential benefits of combining ribitol with AAV gene therapy for treating FKRP-related muscular dystrophy. The fact that ribitol is a metabolite in nature and has already been tested in animal models and clinical trials in humans without severe side effects provides a safety profile for it to be trialed in combination with AAV gene therapy.

Metabolic Reprogramming by Ribitol Expands the Therapeutic Window of BETi JQ1 against Breast Cancer.

Many cancer patients still lack effective treatments, and pre-existing or acquired resistance limits the clinical benefit of even the most advanced medicines. Recently, much attention has been given to the role of metabolism in cancer, expanding from the Warburg effect to highlight unique patterns that, in turn, may improve diagnostic and therapeutic approaches. Our recent metabolomics study revealed that ribitol can alter glycolysis in breast cancer cells. In the current study, we investigate the combinatorial effects of ribitol with several other anticancer drugs (chrysin, lonidamine, GSK2837808A, CB-839, JQ1, and shikonin) in various breast cancer cells (MDA-MB-231, MCF-7, and T-47D). The combination of ribitol with JQ1 synergistically inhibited the proliferation and migration of breast cancer cells cell-type dependently, only observed in the triple-negative MDA-MB-231 breast cancer cells. This synergy is associated with the differential effects of the 2 compounds on expression of the genes involved in cell survival and death, specifically downregulation in c-Myc and other anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1), but upregulation in p53 and cytochrome C levels. Glycolysis is differentially altered, with significant downregulation of glucose-6-phosphate and lactate by ribitol and JQ1, respectively. The overall effect of the combined treatment on metabolism and apoptosis-related genes results in significant synergy in the inhibition of cell growth and induction of apoptosis. Given the fact that ribitol is a metabolite with limited side effects, a combined therapy is highly desirable with relative ease to apply in the clinic for treating an appropriate cancer population. Our results also emphasize that, similar to traditional drug development, the therapeutic potential of targeting metabolism for cancer treatment may only be achieved in combination with other drugs and requires the identification of a specific cancer population. The desire to apply metabolomic intervention to a large scope of cancer types may be one of the reasons identification of this class of drugs in a clinical trial setting has been delayed.

Inhibitory machinery for the functional dystroglycan glycosylation.

Dystroglycan (DG), a muscular transmembrane protein, plays a critical role in transducing extracellular matrix-derived signals to the cytoskeleton and provides physical strength to skeletal muscle cell membranes. The extracellular domain of DG, α-DG, displays unique glycosylation patterns. Fully functional glycosylation is required for this domain to interact with components of extracellular matrices, including laminin. One of the unique sugar compositions found in such functional glycans on DG is two ribitol phosphates that are transferred by the sequential actions of fukutin (FKTN) and fukutin-related protein (FKRP), which use CDP-ribitol as a donor substrate. These are then further primed for matriglycan biosynthesis. A recent in vitro study reported that glycerol phosphate could be similarly added to α-DG by FKTN and FKRP if they used CDP-glycerol (CDP-Gro) as a donor substrate. However, the physiological relevance of these findings remains elusive. Imae et al. addressed the knowledge gap regarding whether CDP-Gro is present in mammals and how CDP-Gro is synthesized and functions in mammals.

An Alternate Process for the Solid-Phase Synthesis and Solid-Phase Purification of Synthetic Nucleic Acid Sequences.

The chemical synthesis of a riboside phosphoramidite has been achieved to provide a 5-O-capture linker and a 2-O-silyl ether protecting group with the intent of enabling an efficient solid-phase purification of synthetic DNA sequences. The riboside phosphoramidite has been incorporated into a DNA sequence while performing the penultimate automated solid-phase synthesis cycle of the sequence. The terminal 5-O-riboside moiety of the resulting DNA sequence is then conjugated to a capture linker to create an anchor for the solid-phase purification of the DNA sequence conjugate. Release of all DNA sequences from the synthesis support is achieved under standard basic conditions to yield a mixture of the desired DNA sequence conjugate along with unconjugated, shorter-than-full-length sequence contaminants. Upon exposure of all DNA sequences to a capture solid support, only the DNA sequence conjugate is chemoselectively captured, thereby allowing the unconjugated shorter-than-full-length DNA sequences to be efficiently washed away from the capture support. After 2-O-cleavage of the silyl ether protecting group from the terminal riboside ethylphosphate triester conjugate, the solid-phase-purified DNA sequence is efficiently released from the capture support through an innovative intramolecular cyclodeesterification of the ethylphosphate triester, prompted by the riboside's rigid cis-diol conformer, to provide a highly pure DNA sequence. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-1,4-anhydro-D-ribitol (3) Basic Protocol 2: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-3-O-[(N,N-diisopropylamino)ethyloxyphosphinyl]-1,4-anhydro-D-ribitol (6). Basic Protocol 3: Automated synthesis of the chimeric solid-phase-linked DNA sequence 8. Support Protocol: Preparation of 2-cyanoethyl-(5-oxohexyl)-N,N-diisopropylphosphoramidite (9). Basic Protocol 4: Solid-phase purification of the chimeric DNA sequence 10.

Molecular size distribution assessment of Haemophilus influenzae vaccine containing lactose by HPAEC-PAD and colorimetric assays.

Molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccine is an important indicator for its immunogenicity and stability. Molecular size distribution was evaluated by High-Performance Protein Chromatography on Sepharose CL-4B column, and fractions were pooled. The use of high flow rate, incorporation of a calibration standard with the injected buffer and pooling method yielded a superior assay compared to conventional pharmacopeial method. The pools were analyzed for determination of distribution coefficient (KD) of 0.2 and 0.5 using two validated techniques: High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC-PAD) for ribitol determination and an optimized colorimetric assay for phosphorus determination. Linearity was achieved over range of 0.10-10.0 µg/mL and 1.0-8.0 µg/mL with LOD of 0.03 and 0.28 µg/mL for HPAEC and colorimetric assays, respectively. The developed assays were successfully applied in quality control monitoring of Hib conjugate vaccine. The optimized colorimetric method had shortened the analysis time to 25 min compared to 3.5 h for the European pharmacopeial assay by modifying the burning process. HPAEC stability results revealed 40% decrease in MSD after stressed storage conditions. The proposed assays offer a reliable and economic platform for monitoring the quality attributes of Hib for biopharma industry.

Dystroglycanopathy: From Elucidation of Molecular and Pathological Mechanisms to Development of Treatment Methods.

Dystroglycanopathy is a collective term referring to muscular dystrophies with abnormal glycosylation of dystroglycan. At least 18 causative genes of dystroglycanopathy have been identified, and its clinical symptoms are diverse, ranging from severe congenital to adult-onset limb-girdle types. Moreover, some cases are associated with symptoms involving the central nervous system. In the 2010s, the structure of sugar chains involved in the onset of dystroglycanopathy and the functions of its causative gene products began to be identified as if they were filling the missing pieces of a jigsaw puzzle. In parallel with these discoveries, various dystroglycanopathy model mice had been created, which led to the elucidation of its pathological mechanisms. Then, treatment strategies based on the molecular basis of glycosylation began to be proposed after the latter half of the 2010s. This review briefly explains the sugar chain structure of dystroglycan and the functions of the causative gene products of dystroglycanopathy, followed by introducing the pathological mechanisms involved as revealed from analyses of dystroglycanopathy model mice. Finally, potential therapeutic approaches based on the pathological mechanisms involved are discussed.

Enhanced culturing techniques for the mycobiont isolated from the lichen Xanthoria parietina.

Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well as methods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, on the growth of the axenically cultured mycobiont isolated from the lichen Xanthoria parietina were tested. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1% D-glucose used in the originally described LBM by either 1%, 2% or 3% of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11557-021-01707-7.

Ribitol in Solution Is an Equilibrium of Asymmetric Conformations.

Ribitol (C5H12O5), an acyclic sugar alcohol, is present on mammalian α-dystroglycan as a component of O-mannose glycan. In this study, we examine the conformation and dynamics of ribitol by database analysis, experiments, and computational methods. Database analysis reveals that the anti-conformation (180°) is populated at the C3-C4 dihedral angle, while the gauche conformation (±60°) is seen at the C2-C3 dihedral angle. Such conformational asymmetry was born out in a solid-state 13C-NMR spectrum of crystalline ribitol, where C1 and C5 signals are unequal. On the other hand, solution 13C-NMR has identical chemical shifts for C1 and C5. NMR 3J coupling constants and OH exchange rates suggest that ribitol is an equilibrium of conformations, under the influence of hydrogen bonds and/or steric hinderance. Molecular dynamics (MD) simulations allowed us to discuss such a chemically symmetric molecule, pinpointing the presence of asymmetric conformations evidenced by the presence of correlations between C2-C3 and C3-C4 dihedral angles. These findings provide a basis for understanding the dynamic structure of ribitol and the function of ribitol-binding enzymes.

(Automated) Synthesis of Well-defined Staphylococcus Aureus Wall Teichoic Acid Fragments.

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.

The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi.

The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency.

Glycoconjugate Vaccines for Prevention of Haemophilus influenzae Type b Diseases.

This review summarizes the experience in laboratory- and industrial-scale syntheses of glycoconjugate vaccines used for prevention of infectious diseases caused by Haemophilus influenzae type b bacteria based on the linear capsular polysaccharide poly-3-ß-D-ribosyl-(1→1)-D-ribitol-5-phosphate (PRP) or related synthetic oligosaccharide ligands. The methods for preparation of related oligosaccharide derivatives and results of the studies evaluating effect of their length on immunogenic properties of the conjugates with protein carriers are overviewed.

ISPD Overexpression Enhances Ribitol-Induced Glycosylation of α-Dystroglycan in Dystrophic FKRP Mutant Mice.

Dystroglycanopathy, a subgroup of muscular dystrophies, is characterized by hypoglycosylation of α-dystroglycan (α-DG), which reduces its laminin-binding activity to extracellular matrix proteins, causing progressive loss of muscle integrity and function. Mutations in the fukutin-related protein (FKRP) gene are the most common causes of dystroglycanopathy. FKRP transfers ribitol-5-phosphate to the O-mannosyl glycan on α-DG from substrate cytidine diphosphate (CDP)-ribitol, which is synthesized by isoprenoid synthase domain-containing protein (ISPD). We previously reported that oral administration of ribitol restores therapeutic levels of functional glycosylation of α-DG (F-α-DG) in a FKRP mutant mouse model. Here we examine the contribution of adeno-associated virus (AAV)-mediated overexpression of ISPD to the levels of CDP-ribitol and F-α-DG with and without ribitol supplementation in the disease model. ISPD overexpression alone and in combination with ribitol improves dystrophic phenotype. Furthermore, the combined approach of ribitol and ISPD acts synergistically, increasing F-α-DG up to 40% of normal levels in cardiac muscle and more than 20% in limb and diaphragm. The results suggest that low levels of substrate limit production of CDP-ribitol, and endogenous ISPD also becomes a limiting factor in the presence of a supraphysiological concentration of ribitol. Our data support further investigation of the regulatory pathway for enhancing efficacy of ribitol supplement to FKRP-related dystroglycanopathy.

Mammalian O-mannosyl glycans: Biochemistry and glycopathology.

Glycosylation is an important posttranslational modification in mammals. The glycans of glycoproteins are classified into two groups, namely, N-glycans and O-glycans, according to their glycan-peptide linkage regions. Recently, O-mannosyl glycan, an O-glycan, has been shown to be important in muscle and brain development. A clear relationship between O-mannosyl glycans and the pathomechanisms of some congenital muscular dystrophies has been established in humans. Ribitol-5-phosphate is a newly identified glycan component in mammals, and its biosynthetic pathway has been elucidated. The discovery of new glycan structures and the identification of highly regulated mechanisms of glycan processing will help researchers to understand glycan functions and develop therapeutic strategies.

Antibodies prevalence against Haemophilus influenzae type b in Jeddah population, Saudi Arabia. II. Antibodies subclasses.

Our previous reported that Jeddah, Saudi Arabia population have a comparable and protective herd immunity represented by IgG, IgA and IgM against Haemophilus influenzae type b (Hib), by using indirect ELISA test was used to evaluate Haemophilus influenzae type b (Hib) anti-polyribosyl-ribitol phosphate (PRP) total antibodies (IgM, IgG, IgA) in 1,003 sera samples. Current report was evaluated the IgG and IgA subclasses responsible about this protection using same methodology. IgG, IgA, and their subclasses are responsible about this circulating protection. Our study will consider the first report evaluate the levels of IgA subclasses and its relation to ages, as well as the relations between anti-Hib antibody subclass and age. The current results demonstrated that the highest levels concentrated in IgG1 and IgG2, while IgG3 and IgG4 showed the lowest levels. So, their concentrations were arranged in IgG1 > IgG2 > IgG3 > IgG4. The results indicated that the age and gender have no effect on both IgG or IgA subclasses in healthy immunized individuals enrolled. While, IgA1 concentrations were significantly higher than IgA2 in all age categories regardless of gender. It seem that the IgG1, IgG2 and IgA1 subclasses were the main constituent of Jeddah herd immunity against Hib. Finally, to the best of our knowledge, there were no previous reports that focusing on the levels of IgA subclasses and its relation to ages, so our study considers the first worldwide.

Antibodies prevalence against Haemophilus influenzae type b in Jeddah population, Saudi Arabia. III. Antibodies avidity.

Our previous results showed higher level of anti-Hib IgG (2.41 µg/ml) average geometric mean concentration (GMC) regardless of age and gender, followed by levels of IgM (0.91 µg/ml) and IgA (0.34 µg/ml), reflecting the prevalence of immunity against Hib. Current study was evaluated the avidity profile for these high anti-Hib concentration. The IgM avidity analysis did not revealed any significant results between male and female values. While, the average of IgA avidity in male samples were significantly (P< 0.05) higher than the average of female. Also, The result showed that high percentage of population has moderate to high IgG avidity. The study showed lower avidity percentages of both IgM and IgA comparing to IgG. More studies are suggested to be done on individuals with low avidity considering the secondary immunodeficiency in participant's history.

Estudo da microfiltração de fluxo tangencial para a clarificação do caldo fermentado de Haemophilus influenzae tipo b

Haemophilus influenzae type b (Hib) is a Gram-negative, facultative aerobic bacteria, responsible for respiratory infections, like pneumonia and meningitis, in children younger than 5 years. The main virulence factor of Hib is its capsular exopolysaccharide, Polyribosyl- Ribitol-Phosphate (PRP). Traditional PRP downstream processing comprises several steps of ethanol precipitation, centrifugation, ultracentrifugation, an also phenol extraction, resulting in a laborious, expensive and complex process. Therefore, a simpler and easily scalable process has been developed using tangential flow filtration (TFF) and ethanol precipitation steps. The aim of this work was to study the influence of the main variables during the microfiltration of the fermented broth for PRP recovery. Cell separation was performed at constant volume of 0.5 L, using a TFF system equipped with a Pellicon 2 Mini PVDF membrane (0.22 μm, 0.1 m2 ) and PBS pH 6.3 as dialysis buffer. Permeate flow was gradually increased during process, starting at 5 L.h-1.m-2 up to established values, in order to avoid membrane fouling. A full factorial experimental design was employed, in which three variables were combined at two levels, namely, TMP (15 and 30 psi), feed flow rate (400 and 500 L.h-1.m-2) and permeate flux (15 and 25 L.h-1.m-2), while temperature (22°C), PRP concentration (1 mg/mL), and broth volume/membrane area ratio (5 L/m2) remained constant. The effect of each variable was statistically evaluated by Analysis of Variance. The results indicate that TMP, feed flow, and permeate flow do not affect PRP retention by the membrane when evaluated independently. On the other hand, when combined on pairs or all simultaneously, these variables changed the PRP retention. The interaction between the higher experimental settings of TMP and feed flow or TMP and permeate flow increased PRP retention by around 8% and consequently reduced PRP recovery. Controlling these parameters is a promising strategy for the optimization of the broth clarification process, since the mean recovery observed in the trials was over 80%, in opposition to a mean recovery of around 60% obtained in former experiments.

Haemophilus influenzae tipo b (Hib) é uma bactéria Gram-negativa, aeróbia facultativa, responsável por infecções respiratórias, como pneumonia e meningite, em crianças menores de 5 anos. O principal fator de virulência do Hib é seu exopolissacarídeo capsular, Poliribosil- Ribitol-Fosfato (PRP). O processamento tradicional do PRP compreende várias etapas de precipitação com etanol, de centrifugação, de ultracentrifugação e de extração com fenol, resultando em um processo trabalhoso, caro e complexo. Desenvolveu-se, portanto, um processo mais simples e facilmente escalonável utilizando diversas etapas de filtração de fluxo tangencial (TFF), combinadas com etapas de precipitação com etanol. O objetivo deste trabalho foi estudar a influência das principais variáveis durante a microfiltração do caldo fermentado para a recuperação do PRP. A separação celular foi realizada a volume constante de 0,5 L, utilizando um sistema TFF equipado com uma membrana Pellicon 2 Mini PVDF (0,22 μm, 0,1 m2 ) e tampão fosfato salino pH 6,3 para a diálise. O fluxo de permeado foi gradualmente aumentado durante o processo, iniciando em 5 L.h-1.m-2 até valores estabelecidos, a fim de evitar o entupimento na membrana. Utilizou-se o delineamento experimental fatorial completo, no qual três variáveis foram combinadas em dois níveis: TMP (15 e 30 psi), fluxo de alimentação (400 e 500 L.h-1.m-2) e fluxo de permeado (15 e 25 L.h- 1 .m-2), enquanto a temperatura (22° C), a concentração de PRP (1 mg/mL) e a relação volume de caldo / área de membrana (5 L/m2) permaneceram constantes. O efeito das variáveis selecionadas foi avaliado estatisticamente pela Análise de Variância. Os resultados indicam que o fluxo de alimentação e o fluxo de permeado não afetam a retenção do PRP pela membrana quando avaliados de forma independente. Por outro lado, quando combinados em pares, essas variáveis alteraram a retenção do PRP. A interação entre as configurações experimentais mais altas de TMP e fluxo de alimentação aumentou a retenção de PRP em cerca de 8% e, consequentemente, reduziu a recuperação de PRP. O controle desses parâmetros é uma estratégia promissora para a otimização do processo de clarificação do caldo, uma vez que a recuperação média observada nos ensaios foi superior a 80%, contra uma recuperação média de cerca de 60% em experimentos anteriores.

Uso de microfiltração tangencial no processo de purificação do exopolissacarídeo capsular de Haemophilus influenzae tipo B

Haemophilus influenzae type b (Hib) is a Gram-negative bacterium, responsible for serious infections, such as pneumonia, sepsis and meningitis, especially in children younger than 5 years old and immunocompromised individuals. The main virulence factor of Hib is its polyribosyl-ribitol-phosphate capsular exopolysaccharide (PRP), which is used in the formulation of Hib vaccines, conjugated to a carrier protein. The production of the conjugated vaccine is complex and costly, particularly the PRP purification process, that comprises several steps of ethanol precipitation and centrifugation, and uses organic solvents and detergents. Targeting a more affordable vaccine that meets the public health systems demands, the Process Development Laboratory of Butantan Institute has been developing a simpler, economical and easily scalable purification process based on tangential flow filtration. In this process, one major bottleneck is the broth clarification step by microfiltration, which results in low PRP recovery and high variability, probably due to membrane fouling. Regarding this matter, a small scale study was previously performed, in order to identify the best operational conditions of relevant process variables, namely transmembrane pressure (TMP), feed flow, and permeate flow. The aim of this work was to scale up the broth clarification step using the defined conditions for these parameters and to perform a critical flux analysis, which is a very useful tool in tangential flow filtration optimization. Although PRP recovery was relatively smaller in the higher scale (68.5% against 97.5%), the permeate flow and TMP profiles were very similar. Analysis of PRP recovery profile indicated that extending dialysis might improve PRP yield. Critical flux of around 35 L.h-1 .m-2 was determined experimentally, which explains the high PRP recovery when a limit permeate flux of 25 L.h-1 .m-2 was employed in the cell clarification step. In conclusion, the process designed for broth clarification is quite promising in terms of product yield and scalability.

Haemophilus influenzae do tipo b (Hib) é uma bactéria Gram-negativa, responsável por infecções graves, como pneumonia, sepse e meningite, especialmente em crianças menores de 5 anos de idade e indivíduos imunocomprometidos. Seu principal fator de virulência é o exopolissacarídeo capsular de poliribosil-ribitol- fosfato (PRP), que é utilizado na formulação de vacinas contra Hib, conjugado a uma proteína carreadora. A produção da vacina conjugada é complexa e onerosa, principalmente o processo de purificação do PRP, caracterizado por várias etapas de precipitação com etanol, centrifugação e uso de solventes orgânicos e detergentes. Buscando uma vacina mais acessível, que possa atender à demanda de sistemas públicos de saúde, o Laboratório de Desenvolvimento de Processos vem desenvolvendo nos últimos anos um método de purificação mais simples, econômico e facilmente escalonável, baseado em filtração tangencial. Nesse processo, um dos gargalos identificados é a etapa de separação celular por microfiltração, que apresentava baixa recuperação de PRP e alta variabilidade dos resultados, provavelmente relacionados ao fenômeno de fouling. Um estudo em pequena escala foi realizado previamente para identificar as melhores condições de variáveis operacionais relevantes - pressão transmembrana (TMP), fluxo de entrada e fluxo do filtrado. O presente trabalho teve como objetivos escalonar o processo, mantendo essas condições, e realizar uma análise do fluxo crítico do filtrado, ferramenta bastante útil para a otimização da filtração tangencial. Embora a recuperação de PRP tenha sido relativamente menor no processo escalonado (68,5% contra 97,5%), os perfis de fluxo do filtrado e de TMP se mostraram muito semelhantes, e as análises da tendência de recuperação mostram que um aumento no número de diavolumes utilizado pode contornar essa questão. O valor de fluxo crítico determinado experimentalmente foi próximo de 35 L.h-1 .m-2 , o que corrobora os bons resultados obtidos com o uso de fluxo limite de 25 L.h-1 .m-2 para o filtrado no processo de separação celular. Os resultados obtidos mostram que o desenho de processo estabelecido para a clarificação de PRP é bastante promissor em termos de rendimento de PRP e escalabilidade do processo.