Process development for production and purification of the Schistosoma mansoni Sm14 antigen.

The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+ transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 °C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250  mg L-1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.

Development and characterization of bio-derived polyhydroxyalkanoate nanoparticles as a delivery system for hydrophobic photodynamic therapy agents.

In this study, we developed and investigated nanoparticles of biologically-derived, biodegradable polyhydroxyalkanoates (PHAs) as carriers of a hydrophobic photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H, 23H-porphine (pTHPP) for photodynamic therapy (PDT). Three PHA variants; polyhydroxybutyrate, poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-HV) with 12 and 50% HV were used to formulate pTHPP-loaded PHA nanoparticles by an emulsification-diffusion method, where we compared two different poly(vinyl alcohol) (PVA) stabilizers. The nanoparticles exhibited nano-scale spherical morphology under TEM and hydrodynamic diameters ranging from 169.0 to 211.2 nm with narrow size distribution. The amount of drug loaded and the drug entrapment efficiency were also investigated. The in vitro photocytotoxicity was evaluated using human colon adenocarcinoma cell line HT-29 and revealed time and concentration dependent cell death, consistent with a gradual release pattern of pTHPP over 24 h. This study is the first demonstration using bacterially derived P(HB-HV) copolymers for nanoparticle delivery of a hydrophobic photosensitizer drug and their potential application in PDT.

Secreted production of assembled Norovirus virus-like particles from Pichia pastoris.

BACKGROUND: Norovirus virus-like particles (NoV VLPs) have recently been explored as potential vaccine platforms due to their ability to produce an effective immune response. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. These NoV VLPs have been expressed in Escherichia coli, yeast and insect cells. Expression in E. coli and insect cells share downstream processing issues due to the presence of inclusion bodies or the need for numerous purification steps. NoV VLPs have also been produced in the yeast P. pastoris; however the protein was only expressed intracellularly. RESULTS: We have successfully expressed and secreted the VP1 protein in the novel P. pastoris strain, Bg11, using the methanol inducible pJ912 expression vector, containing the cDNA of NoV VP1. Expression of the VP1 protein in Bg11 was carried out in a 1.5 L bioreactor resulting in a total yield of NoV VLPs greater than 0.6 g/L. NoV VLPs obtained from the culture supernatant were purified via ion-exchange chromatography, resulting in particles with a purity over 90%. The average size of the particles after purification was 40 nm. Transmission electron microscopy was used to visualize the morphology of the particles and saliva-binding assay confirmed that the NoV VLPs bind to Histo-Blood Group Antigens (HBGA). CONCLUSIONS: In this study we describe the expression and characterization of fully assembled Norovirus virus-like particles obtained from P. pastoris. The particles are similar in size, morphology and binding capacity, as previously described, for the original NoV. Our results detail the successful expression and secretion of VLPs in P. pastoris, improving their candidacy as a vaccine platform.

Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591.

A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications.

Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.

Targeted near-IR hybrid magnetic nanoparticles for in vivo cancer therapy and imaging.

We report the use of immuno-targeted gold-iron oxide hybrid nanoparticles for laser-assisted therapy and for MRI-based imaging as demonstrated in xenograft colorectal cancer tumor model. Immuno-targeted gold-iron oxide nanoparticles selectively accumulate in SW1222 xenograft tumors as compared to the accumulation in non-antigen-expressing tumor xenografts. Effective photothermal treatment using near-IR laser irradiation (808nm, 5W cm(-2)) application is shown where >65% of the antigen-expressing tumor cells presented corrupt extracellular matrix and cytoplasmic acidophilia suggesting effectiveness of nanoparticle-assisted thermal therapy. Cell killing was confirmed by hematoxylin and eosin (H&E) histological staining where scar-like structure containing collagen bundles was observed in the treatment group. Further, systemically injected HNPs were shown to be effective T2 magnetic resonance (MR) imaging contrast agents, localized and detected at the antigen-expressing xenograft tumors. These findings suggest that the new class of bio-conjugated HNPs exhibits great potential for dual-therapy and diagnostics (theranostics) applications. FROM THE CLINICAL EDITOR: This team reports the successful use of immuno-targeted gold-iron oxide hybrid nanoparticles for both laser-assisted therapy and MRI-based imaging in a xenograft colorectal cancer tumor model, demonstrating strong potentials for dual applications in cancer diagnosis and therapy.

Paradoxical glomerular filtration of carbon nanotubes.

The molecular weight cutoff for glomerular filtration is thought to be 30-50 kDa. Here we report rapid and efficient filtration of molecules 10-20 times that mass and a model for the mechanism of this filtration. We conducted multimodal imaging studies in mice to investigate renal clearance of a single-walled carbon nanotube (SWCNT) construct covalently appended with ligands allowing simultaneous dynamic positron emission tomography, near-infrared fluorescence imaging, and microscopy. These SWCNTs have a length distribution ranging from 100 to 500 nm. The average length was determined to be 200-300 nm, which would yield a functionalized construct with a molecular weight of approximately 350-500 kDa. The construct was rapidly (t(1/2) approximately 6 min) renally cleared intact by glomerular filtration, with partial tubular reabsorption and transient translocation into the proximal tubular cell nuclei. Directional absorption was confirmed in vitro using polarized renal cells. Active secretion via transporters was not involved. Mathematical modeling of the rotational diffusivity showed the tendency of flow to orient SWCNTs of this size to allow clearance via the glomerular pores. Surprisingly, these results raise questions about the rules for renal filtration, given that these large molecules (with aspect ratios ranging from 100:1 to 500:1) were cleared similarly to small molecules. SWCNTs and other novel nanomaterials are being actively investigated for potential biomedical applications, and these observations-that high aspect ratio as well as large molecular size have an impact on glomerular filtration-will allow the design of novel nanoscale-based therapeutics with unusual pharmacologic characteristics.

Protein secretion in Pichia pastoris and advances in protein production.

Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production.

Norovirus-VLPs expressing pre-erythrocytic malaria antigens induce functional immunity against sporozoite infection.

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.

Tumor-specific hybrid polyhydroxybutyrate nanoparticle: surface modification of nanoparticle by enzymatically synthesized functional block copolymer.

A new approach to functionalize the surface of hydrophobic nanocarrier through enzymatic polymerization was demonstrated. The effective coupling between the hydrophobic surface of PHB nanoparticle and PHB chain grown from the enzyme fused with a specific ligand provided a simple way of functionalizing nanoparticles with active protein layers in aqueous environment. PHB nanoparticles loaded with model drug molecule, Nile red, were prepared through oil-in-water emulsion solvent evaporation method and the surface of nanoparticles were functionalized with tumor-specific ligand, RGD4C, fused with PHA synthase that drove the coupling reaction. The functionalized PHB nanoparticles showed a specific affinity to MDA-MB 231 breast cancer cells indicating that the tumor-specific ligand, RGD4C, was effectively displayed on the surface of PHB nanoparticles through enzymatic modification and confers targeting capability on the drug carrier.

A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures.

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.

Low-power microfluidic electro-hydraulic pump (EHP).

Low-power electrolysis-based microfluidic pumps utilizing the principle of hydraulics, integrated with microfluidic channels in polydimethylsiloxane (PDMS) substrates, are presented. The electro-hydraulic pumps (EHPs), consisting of electrolytic, hydraulic and fluidic chambers, were investigated using two types of electrodes: stainless steel for larger volumes and annealed gold electrodes for smaller-scale devices. Using a hydraulic fluid chamber and a thin flexible PDMS membrane, this novel prototype successfully separates the reagent fluid from the electrolytic fluid, which is particularly important for biological and chemical applications. The hydraulic advantage of the EHP device arises from the precise control of flow rate by changing the electrolytic pressure generated, independent of the volume of the reagent chamber, mimicking the function of a hydraulic press. Since the reservoirs are pre-filled with reagents and sealed prior to testing, external fluid coupling is minimized. The stainless steel electrode EHPs were manufactured with varying chamber volume ratios (1 : 1 to 1 : 3) as a proof-of-concept, and exhibited flow rates of 1.25 to 30 microl/min with electrolysis-based actuation at 2.5 to 10 V(DC). The miniaturized gold electrode EHPs were manufactured with 3 mm diameters and 1 : 1 chamber volume ratios, and produced flow rates of 1.24 to 7.00 microl/min at 2.5 to 10 V(AC), with a higher maximum sustained pressure of 343 KPa, suggesting greater device robustness using methods compatible with microfabrication. The proposed technology is low-cost, low-power and disposable, with a high level of reproducibility, allowing for ease of fabrication and integration into existing microfluidic lab-on-a-chip and analysis systems.

Multiplex single nucleotide polymorphism genotyping utilizing ligase detection reaction coupled surface enhanced Raman spectroscopy.

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity.

Gold hybrid nanoparticles for targeted phototherapy and cancer imaging.

Gold and iron oxide hybrid nanoparticles (HNPs) synthesized by the thermal decomposition technique are bio-functionalized with a single chain antibody, scFv, that binds to the A33 antigen present on colorectal cancer cells. The HNP-scFv conjugates are stable in aqueous solution with a magnetization value of 44 emu g(-1) and exhibit strong optical absorbance at 800 nm. Here we test this material in targeting, imaging and selective thermal killing of colorectal cancer cells. Cellular uptake studies showed that A33-expressing cells take up the A33scFv-conjugated HNPs at a rate five times higher than cells that do not express the A33 antigen. Laser irradiation studies showed that approximately 53% of the A33-expressing cells exposed to targeted HNPs are killed after a six-minute laser treatment at 5.1 W cm(-2) using a 808 nm continuous wave laser diode while < 5% of A33-nonexpressing cells are killed. At a higher intensity, 31.5 W cm(-2), the thermal destruction increases to 99 and 40% for A33-expressing cells and A33 nonexpressing cells, respectively, after 6 min exposure. Flow cytometric analyses of the laser-irradiated A33 antigen-expressing cells show apoptosis-related cell death to be the primary mode of cell death at 5.1 W cm(-2), with increasing necrosis-related cell death at higher laser power. These results suggest that this new class of bio-conjugated hybrid nanoparticles can potentially serve as an effective antigen-targeted photothermal therapeutic agent for cancer treatment as well as a probe for magnetic resonance-based imaging.

Recombinant immunotherapeutics: current state and perspectives regarding the feasibility and market.

Recombinant immunotherapeutics are important biologics for the treatment and prevention of various diseases. Immunotherapy can be divided into two categories, passive and active. For passive immunotherapy, the successes of antibody and cytokine therapeutics represent a promising future and opportunities for improvements. Efforts, such as cell engineering, antibody engineering, human-like glycosylation in yeast, and Fab fragment development, have led the way to improve antibody efficacy while decreasing its high manufacturing costs. Both new cytokines and currently used cytokines have demonstrated therapeutic effects for different indications. As for active immunotherapy, recently approved HPV vaccines have encouraged the development of preventative vaccines for other infectious diseases. Immunogenic antigens of pathogenic bacteria can now be identified by genomic means (reverse vaccinology). Due to the recent outbreaks of pandemic H1N1 influenza virus, recombinant influenza vaccines using virus-like particles and other antigens have also been engineered in several different recombinant systems. However, limitations are found in existing immunotherapeutics for cancer treatment, and recent development of therapeutic cancer vaccines such as MAGE-A3 and NY-ESO-1 may provide alternative therapeutic strategy.

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal.

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L(-1). Decreasing the cultivation temperature from 25 degrees C to 16 degrees C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL(-1), and the yield of SSX2NORD was 21.6 mg L(-1).

Surface-enhanced Raman scattering based ligase detection reaction.

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.

Detection of carbendazim by surface-enhanced Raman scattering using cyclodextrin inclusion complexes on gold nanorods.

Surface-enhanced Raman scattering (SERS) was used for the detection of carbendazim using gold nanorods derivatized with a beta-cyclodextrin derivative to bind this fungicide. Gold nanorods were synthesized with an aspect ratio of 3.3 to match the 785 nm excitation wavelength used in the SERS studies. A thiolated cyclodextrin-based sensor molecule was synthesized, and its inclusion complex with carbendazim was formed and studied using SERS spectroscopy. SERS analysis of the inclusion complex at different concentrations in the presence of gold nanorods afforded good quality Raman spectra of carbendazim at micromolar concentrations. Quantitative analysis was preformed using partial least-squares (PLS), and a calibration plot for these data was generated. Results suggest that carbendazim concentrations as low as 50 microM can be accurately detected using the described SERS assay.

Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography.

Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.

High-level expression of a phage display-derived scFv in Pichia pastoris.

Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification of A33scFv was optimized for the methylotrophic yeast P. pastoris. P. pastoris with a Mut(S) phenotype was selected to express A33scFv under regulation of the methanol-inducible AOX1 promoter. Here we describe a large-scale fed-batch fermentation process with an efficient online closed-loop methanol control for the production of the recombinant protein. Purification of A33scFv from clarified culture medium was done using a two-step chromatographic procedure using anion exchange and hydrophobic interaction chromatography, resulting in a final product with more than 90% purity. This chapter provides protocols that can be used as a base for process development of recombinant protein expression in P. pastoris and purification of these proteins for use in further functionality studies and in diagnostic and therapeutic applications.