Purification of singly PEGylated α-lactalbumin using charged ultrafiltration membranes

One of the challenges in producing a PEGylated therapeutic protein is that the PEGylation reaction typically generates a mixture of both singly and multiply PEGylated species. The objective of this study was to examine the feasibility of using ultrafiltration for the purification of a singly PEGylated protein from the multiply PEGylated conjugates. Data were obtained with α‐lactalbumin that was PEGylated with a 20 kDa activated PEG, with the ultrafiltration performed over a range of pH and ionic strength using both unmodified and negatively charged composite regenerated cellulose membranes. Purification of the singly PEGylated α‐lactalbumin from the multiply PEGylated species was accomplished using a diafiltration process with a negatively charged membrane at pH 5 and an ionic strength of 0.4 mM, conditions that maximized the electrostatic exclusion of the multiply PEGylated species from the charged membrane. The diafiltration process provided more than 97% yield with greater than 20‐fold purification between the singly and doubly PEGylated proteins and nearly complete removal of the more heavily PEGylated species. The singly PEGylated α‐lactalbumin was recovered as a dilute filtrate solution, although this dilution could be eliminated using a cascade filtration or the final product could be re‐concentrated in a second ultrafiltration as part of the final formulation. These results demonstrate the feasibility of using ultrafiltration for the purification of singly PEGylated protein therapeutics. Biotechnol. Bioeng. 2011; 108:822–829. © 2010 Wiley Periodicals, Inc.     

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨?KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1012–1018, 2012     

Intermolecular interactions during ultrafiltration of pegylated proteins

Recent studies have demonstrated the feasibility of using membrane ultrafiltration for the purification of pegylated proteins; however, the separations have all been performed at relatively low protein concentrations where intermolecular interactions are unimportant. The objective of this study was to examine the behavior at higher PEG concentrations and to develop an appropriate theoretical framework to describe the effects of intermolecular interactions. Ultrafiltration experiments were performed using pegylated α‐lactalbumin as a model protein with both neutral and charged composite regenerated cellulose membranes. The transmission of the pegylated α‐lactalbumin, PEG, and α‐lactalbumin all increase with increasing PEG concentration due to the increase in the solute partition coefficient arising from unfavorable intermolecular interactions in the bulk solution. The experimental results were in good agreement with a simple model that accounts for the change in Gibbs free energy associated with these intermolecular interactions, including the effects of concentration polarization on the local solute concentrations upstream of the membrane. These intermolecular interactions are shown to cause a greater than expected loss of pegylated product in a batch ultrafiltration system, and they alter the yield and purification factor that can be achieved during a diafiltration process to remove unreacted PEG. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:655–663, 2013     

Purification of capsular polysaccharide produced by <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> type b through a simple,efficient and suitable method for scale-up

Haemophilus influenzae type b, an encapsulated bacterium, causes meningitis in infants worldwide. The capsular polysaccharide conjugated to a carrier protein is effective in the prevention of such infections. The traditional purification process of polysaccharide from bacterial cultures for vaccine production is based on several selective precipitations with solvents such as: ethanol, phenol, and cationic detergents. The separations of solid and liquid phases are based on continuous centrifugation in explosion proof installations. The lipopolysaccharides are separated by ultracentrifugation. A simple and efficient method that can easily be scaled-up was developed for purification of polysaccharides. The ethanol precipitation was reduced to only two steps. The phenol treatment was substituted by ultrafiltration and enzymatic digestion. Lipopolysaccharide was removed by ultrafiltration together with addition of detergent and chelating agent.     

Affinity ultrafiltration: effects of ligand binding on selectivity and process optimization.

The effective design of affinity ultrafiltration processes using a selective macroligand requires a detailed understanding of the effects of ligand-binding interactions on product yield and purification. Theoretical calculations were performed to evaluate the performance of affinity diafiltration separations with both competitive and independent binding interactions for the product and impurity. The intrinsic selectivity for independent binding decreased during the diafiltration due to the increase in fractional impurity binding as the impurity is selectively removed. The opposite behavior was seen for competitive binding because the strongly bound product displaces the impurity from the binding sites. Purification-yield diagrams were used to examine the effects of affinity-ligand concentration and binding constants on the separation. Model calculations were in excellent agreement with experimental data for the separation of tryptophan isomers using bovine serum albumin as the steroselective macroligand. Simulations with a fixed number of diavolumes show a clear optimum in product yield and purification factor at an intermediate ligand concentration due to the competing effects of the intrinsic selectivity and the rate of impurity removal. These results provide an appropriate framework for the design and optimization of affinity ultrafiltration systems.     

A novel Enzyme-Linked Immuno-Sorbent Assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b–Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib–TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive.     

Experimental design to optimize an <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates

Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (K_D) of native and activated polysaccharides. The resulting data showed that the K_D value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.     

A群脑膜炎球菌多糖-破伤风类毒素结合物中游离多糖测定方法的建立

本文建立一种测定A群脑膜炎球菌多糖－破伤风类毒素（PS－TT）结合物中游离多糖的方法。用80％乙醇使PS－TT结合物沉淀,再用60％乙醇洗涤沉淀使未结合多糖溶解,使与TT结合的和未结合的多糖分离并分别测定。该方法可有效分离结合的与未结合的多糖并分别测定,结合物中外加多糖可定量回收。本方法适用于测定A群脑膜炎球菌多糖－破伤风类毒素结合物中游离多糖含量。     

Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.     

Identification of glycosylated regions in pneumococcal PspA conjugated to serotype 6B capsular polysaccharide

Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.     

The development and characterization of an E. coli O25B bioconjugate vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

     

Production of glycoprotein vaccines in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.     

Separation of protein charge variants by ultrafiltration

The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine epsilon-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.     

Purification of Cas9—RNA complexes by ultrafiltration

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.     

Effect of electrostatic interactions on the ultrafiltration behavior of charged bacterial capsular polysaccharides

Charged polysaccharides are used in the food industry, as cosmetics, and as vaccines. The viscosity, thermodynamics, and hydrodynamic properties of these charged polysaccharides are known to be strongly dependent on the solution ionic strength because of both inter‐ and intramolecular electrostatic interactions. The goal of this work was to quantitatively describe the effect of these electrostatic interactions on the ultrafiltration behavior of several charged capsular polysaccharides obtained from Streptococcus pneumoniae and used in the production of Pneumococcus vaccines. Ultrafiltration data were obtained using various Biomax? polyethersulfone membranes with different nominal molecular weight cutoffs. Polysaccharide transmission decreased with decreasing ionic strength primarily because of the expansion of the charged polysaccharide associated with intramolecular electrostatic repulsion. Data were in good agreement with a simple theoretical model based on solute partitioning in porous membranes, with the effective size of the different polysaccharide serotypes evaluated using size exclusion chromatography at the same ionic conditions. These results provide fundamental insights and practical guidelines for exploiting the effects of electrostatic interactions during the ultrafiltration of charged polysaccharides. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1531–1538, 2016     

The purification of recombinant hepatitis B surface antigen by hollow fibre ultrafiltration membrane diafiltration

Summary Recombinant hepatitis B surface antigen (HBsAg) broth was purified by diafiltration with a hollow fibre ultrafiltration membrane (cut off 100 kDa), by pre—treating the HBsAg broth with enzyme, most of the contaminating proteins was removed and very high recovery ratio of HBsAg was achieved. When HBsAg solution was concentrated using hollow fibre ultrafiltration membrane to 10% of its original volume, the HBsAg was recovered almost completely. Accordingly, membrane purification of HBsAg is a high yield, fast method.     

Fusarium verticillioides (Saccardo) Nirenberg Associated with Hardlock of Cotton

The development of new immune potentiators for human vaccines is an important and expanding field of research. In the present study, the ability of the capsular polysaccharide from Neisseria meningitidis serogroup A (CPS-A), a mannose-containing carbohydrate, to enhance the antibody production against a co-administered model vaccine antigen, is examined. A protein-meningococcal serogroup C capsular polysaccharide (CPS-C) conjugate was selected as the model antigen for this study. After subcutaneous immunization of Balb/C mice, the conjugate mixed with CPS-A induced higher anti-CPS-C IgG and IgG_2a antibody levels and higher anti-meningococcal serogroup C bactericidal titers than the conjugate alone or mixed with CPS-C. The immuno-stimulatory properties exhibited by CPS-A and the fact that vaccines based on purified CPS-A has been safely used during decades to fight the serogroup A meningococcal disease, support the proposal to use CPS-A as immune potentiator for human vaccination studies.     

Molecular modeling provides insights into the loading of sialic acid‐containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM₁₉₇. However, not only is there a surprisingly low limit for antigen chain attachment to CRM₁₉₇, but different serogroup saccharides have consistently different CRM₁₉₇ loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW?>?NmC?>?NmY) and the number of chains attached to CRM₁₉₇, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.