Barry Lorbetskie

Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences.

Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.

Optimization and qualification of a quantitative reversed-phase HPLC method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays.

A previously described reversed-phase HPLC (RP-HPLC) method based on fast separations on a non-porous silica stationary phase [1] was optimized and qualified for the quantitative determination of hemagglutinin (HA) in influenza vaccine preparations. Optimization of the gradient elution conditions led to improved separation of the HA1 subunit from other vaccine constituents. The sensitivity of the method was significantly increased by using native fluorescence detection, resulting in an approximately 10-fold increase as compared to UV-vis detection. This enabled the elimination of the concentration step described in the original method and allowed direct analysis of vaccine preparations. The method was qualified for linearity, range, limit of detection, limit of quantitation and precision. Overall, it was found to be linear over the range of 2.5-100 μg HA/mL for all subtypes examined. This range covered 50-150% of the concentration found for individual strains in seasonal influenza vaccines and in the pandemic H1N1 vaccine. The limit of detection and limit of quantitation for each subtype were found to be suitable for the methods intended purpose and compared well to values found by the single radial immunodiffusion (SRID). The repeatability of the method gave RSD values below 5% for both retention time and peak areas. As expected for intermediate precision, larger RSD values for peak area were obtained but were below 10% and deemed acceptable. The RP-HPLC results were compared to Western blot analysis using a HA universal antibody for a set of 15 monovalent A/California H1N1 preparations and showed good correlation. Similarly, the quantitative nature of the RP-HPLC method was assessed in relation to the SRID assay currently used for the determination of the HA content in bulk antigen and final vaccine preparations. Thus, for a series of 23 monovalent A/Brisbane/59/2007 H1N1 bulks, ranging between 12.7 and 15.9 μg HA/mL by SRID, the RP-HPLC values were found to be in very good agreement, ranging between 11.9 and 14.1 μg HA/mL (n=5) for five determinations carried out on 5 different days. During the 2009-10 H1N1 influenza pandemic the quantitative RP-HPLC method was used alongside several other test methods for the analysis of pandemic H1N1 vaccine preparations that included bulk antigen and final vaccines. The HA content of vaccines formulated at 15 or 30 μg/mL was measured by RP-HPLC and SRID and results showed that the HA content determined by RP-HPLC correlated well to that determined by SRID and to values determined by Western blot. Overall, the results provided further evidence of the usefulness of RP-HPLC for the detection and quantitation of the HA content once a reference standard has been established.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry.

A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.

Study of aggregation, denaturation and reduction of interferon alpha-2 products by size-exclusion high-performance liquid chromatography with fluorescence detection and biological assays

Interferon alpha-2 (IFN alpha-2) products have been widely used as antivirals for the treatment of serious diseases such as hepatitis B and C. However, reports of adverse reactions following treatment have prompted investigations into the cause of these undesirable events. In this study size-exclusion HPLC (SE-HPLC) methods coupled with intrinsic fluorescence detection were developed for evaluating the stability and degradation profiles of IFN alpha-2 drug substances and drug products. The method allowed baseline resolution of the active ingredient from the excipients present in the finished products that included large amounts of albumin. Limits of detection (S/N>or=3) for IFN alpha-2a and IFN alpha-2b were 32 ng/mL and 28 ng/mL, respectively and good repeatability of chromatographic profiles (%RSD<2.1) was obtained. High molecular weight (HMW) aggregates with apparent molecular weight of approximately 650 kDa as well as dimers, denatured and reduced variants were successfully identified and separated from native IFN alpha-2 proteins. This chromatographic method, which quantitatively measures physical and chemical changes taking place in solution formulations, was found to be capable of monitoring IFN alpha-2a and IFN alpha-2b stability. Potency assay results revealed up to 87% decrease in biological activity of the physically and chemically altered variants compared to the original IFNs.

Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method

Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

Nanoparticle size and production efficiency are affected by the presence of fatty acids during albumin nanoparticle fabrication

We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.

Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems

Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209–290). Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)—Gen. Subj. 1830(12) (2013) 5515–5525; Kobayashi, Biologicals 34(1) (2006) 55–59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661–4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.