Octanorm [cutaquig®], a new immunoglobulin (human) subcutaneous 16.5% solution for injection (165 mg/mL) - Biochemical characterization, pathogen safety, and stability.

Octanorm (marketed as cutaquig® in Canada and US [2018] and registered in several European countries [2019]) is a new immunoglobulin subcutaneous 16.5% liquid for the treatment of patients with primary immune deficiency (PID) and secondary immune deficiency (SID) depending on country's specific indications. Octanorm contains ≥96% human IgG and is characterized by especially low concentrations of polymers and aggregates, IgA and IgM, a physiological osmolality along with a low isoagglutinin titer. The Octanorm manufacturing process is based on the well-established IVIG octagam® 5% and 10% process, but yields a higher immunoglobulin concentration of 16.5% in the final product. Octanorm shows a distribution of immunoglobulin G subclasses closely proportional to native human plasma and comprises a broad spectrum of antibodies against infectious agents. Potential procoagulant activity is not detectable. IgG functionality and physico-chemical integrity have been demonstrated by state-of-the-art-methods. The virus safety of Octanorm is ensured via a combination of three validated independent methods as part of the manufacturing process. Substantial prion depletion during the manufacturing process has also been demonstrated. Compared with other commercially available subcutaneous immunoglobulin (SCIG) 20% products, Octanorm 16.5% shows a lower viscosity, which is a valuable feature that allows for a more comfortable infusion experience.

Biochemical characterization, stability, and pathogen safety of a new fibrinogen concentrate (fibryga®).

Fibryga® is a new lyophilized fibrinogen concentrate for intravenous use for the treatment of congenital fibrinogen deficiency. fibryga® is produced from pooled human plasma and the final product is characterized by high purity, integrity, and pathogen safety. Functional activity of fibrinogen was demonstrated by cross-linking studies and thromboelastometry; integrity of the fibrinogen molecule was demonstrated by size exclusion chromatography and the detection of only trace amounts of activation markers in the final product. Pathogen safety of fibryga® was proved by downscaling studies for the two dedicated pathogen inactivation/removal steps, i.e. solvent detergent treatment and nanofiltration. Fibryga® is stable for at least three years when stored at room temperature. In conclusion, the performed studies demonstrated that fibryga® meets the requirements for a state-of-the-art fibrinogen concentrate, such as a satisfactory activity profile combined with a favorable pathogen safety profile and stability.

Thawing of Pooled, Solvent/Detergent-Treated Plasma octaplasLG®: Validation Studies Using Different Thawing Devices.

BACKGROUND: The aim of this study was to perform validation of the thawing process for solvent/detergent-treated plasma octaplasLG® using different thawing devices. Optimized settings for temperature and thawing time should be defined based on the results of both temperature measurements and extensive biochemical characterization studies. METHODS: octaplasLG units were thawed using water bath systems (i.e. MB-13A, QuickThaw® DH4), dry tempering systems (i.e. plasmatherm, SAHARA-III), and microwave oven (i.e. transfusio-therm® 2000). Optimized thawing conditions were defined. Subsequently, using the selected thawing conditions, octaplasLG units were thawed and tested on product release parameters. RESULTS: The fastest thawing was observed for the microwave oven. All octaplasLG units thawed by different devices and optimized thawing conditions were clear and free of solid and gelatinous particles, indicating no protein denaturation or overheating. In addition, no significant differences were found in the coagulation and inhibition activity and hemostatic potency of octaplasLG when thawed by the different devices tested. All parameters after thawing were within the product release specification levels. CONCLUSION: Our study demonstrated that octaplasLG can be thawed using all above listed devices without any negative influence on the plasma quality, presupposed that optimized settings defined for this plasma product are used.

Biochemical characterization and stability of immune globulin intravenous 10% liquid (Panzyga®).

Panzyga® is a new glycine-formulated immune globulin intravenous 10% liquid for the treatment of patients suffering from immunodeficiencies and autoimmune diseases. Panzyga® is a high purity, native and functional IgG product with an IgG subclass distribution equivalent to normal plasma. The levels of hemagglutinins and accompanying plasma proteins (including IgA and IgM) are low. Potential procoagulant activity is not detectable. Functional activity of the IgG was demonstrated by opsonophagocytosis and receptor binding assays. Dynamic light scattering measurements and fluorescent dye binding were used to characterize the integrity of the IgG molecule. Panzyga® is stable under refrigerated storage for at least two years regarding all assessed physicochemical and functional parameters; it can also be stored at room temperature for at least twelve months within its total shelf-life.

Targeted profiling of atherogenic phospholipids in human plasma and lipoproteins of hyperlipidemic patients using MALDI-QIT-TOF-MS/MS.

OBJECTIVES: Phospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups. METHODS: Plasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters. RESULTS: Most characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of ∼2-20 µmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = -0.910; p = 0.096) was observed. CONCLUSIONS: Our study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future.

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry.

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24h was inhibited by 95-100% in the presence of HSA but only 0-34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.

A retrospective study of Octaplex in the treatment of bleeding in patients with haemophilia A complicated by inhibitors.

The nonactivated prothrombin complex concentrate (PCC) Octaplex (Octapharma PPGmbH, Vienna, Austria) has been used successfully for the treatment of congenital and acquired coagulation factor deficiencies and associated bleeding. The aims of this study were to assess retrospectively whether Octaplex is an effective treatment option for haemophilia A patients with high-titre inhibitors of factor VIII (FVIII) and to investigate the impact of Octaplex on thrombin generation in vitro and ex vivo. Retrospective data were collected from 15 haemophilia A patients with FVIII inhibitors who had been treated with Octaplex. Mild bleeds were treated for a median of 1 day with a median dose of 77 IU/kg and moderate bleeds for 3 days with 57 IU/kg. The physician's overall satisfaction with Octaplex, taking into account efficacy, safety and cost in comparison with other treatment options, was assessed for each bleed. The overall rating was good, very good or excellent for 29 of 41 (71%) bleeds. No adverse drug reactions were reported. In in-vitro studies of thrombin generation with normal plasma samples, experimental inhibition of FVIII activity prolonged the lag phase, diminished the peak thrombin concentration and decreased the area under the concentration-time curve, as expected. Marked improvement in thrombin generation parameters was achieved by adding 0.5-3 IU factor IX/ml PCC into the samples. The same held true when using plasma samples from haemophilia A patients with FVIII inhibitors. These results demonstrate that Octaplex overcomes inhibition of FVIII in in-vitro and ex-vivo assays of thrombin generation, and that Octaplex is an effective treatment option for haemophilia A patients with FVIII inhibitors.

Thrombin generation capacity is impaired in methylene-blue treated plasma compared to normal levels in single-donor fresh-frozen plasma, a licensed solvent/detergent-treated plasma (Octaplas) and a development product (Uniplas).

Impaired capacity of thrombin generation (TG) was found in single-donor fresh-frozen plasma (FFP) units subjected to medical device treatment by a combination of methylene-blue dye and subsequent white-light exposure (MB plasma, MBP) compared to normal levels in non-MB-treated single-donor FFP, the licensed plasma product Octaplas, and a product under development (Uniplas; working title) applicable independently from the recipient's blood group. This result held true for MBP units obtained from two different European sources revealing a more significant TG impairment using the local inactivation system in blood banks ("in-house") than an industrial MB treatment. Supplementation of functional fibrinogen to physiological levels did not normalise the altered TG capacity in MBP, whereas addition of Octaplas did. No clear-cut correlation between coagulation factor levels in MBP and hampered TG capacity was found, suggesting a composite effect of impairment. A thorough elucidation and evaluation regarding the possible clinical impact of these findings seems prudent.

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry.

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.

Exact molecular mass determination of various forms of native and de-N-glycosylated human plasma-derived antithrombin by means of electrospray ionization ion trap mass spectrometry.

Human plasma-derived antithrombin was characterized in both the native and de-N-glycosylated forms (without separation of isoforms) by means of electrospray ionization ion trap mass spectrometry (ESI-ITMS). In order to determine the limits of the instrument set-up, the molecular mass precision and accuracy of the ESI-ITMS analysis was evaluated with the standard protein enolase and some instrumental data acquisition parameters were optimized. Mass precision was determined as a function of the number of averaged mass spectra (= scans) and data acquisition time. For this study, 20 and 50 scans were averaged and the data acquisition time was chosen to be between 0.5 and 5 min. It turned out that data acquisition times longer than approximately 2 min show no significant differences of the standard deviation of the determined molecular mass. Furthermore, the ion trap scan rate was varied at constant acquisition time of 2 min and the number of averaged scans was set to 20. At the scan rate of 13,000 u s(-1) a mass precision of +/-1.8 Da and a mass accuracy of +0.026% were determined. On reducing the scan rate to 5500 u s(-1), better agreement with the theoretical molecular mass was obtained, showing a mass accuracy of +0.012% but with a decrease in the mass precision to +/-3.0 Da. Using the optimized scan rate of 13,000 u s(-1) and a data acquisition time of 2 min, the exact molecular mass was determined of the three forms of antithrombin, namely the alpha-form, the beta-form and the natural mixture (present in human plasma) containing both forms. The protonated molecular masses were found to be 57,854 and 55,664 Da for the affinity chromatography-isolated alpha-and beta-form, respectively. The mass difference of 2190 Da is attributed to the known difference in carbohydrate content at one specific site. The protonated molecular mass of the dominating species of the natural mixture in human plasma was shown to be 57,850 Da, corresponding to the alpha-form, the major component in native plasma. In this mixture the beta-form was also detected, exhibiting a protonated molecular mass of 55,655 Da, but showing a much lower abundance, as expected. To obtain a complete release of the N-glycan residues by means of PNGase F, a denaturation, reduction and alkylation step of the glycoproteins was performed before the enzymatic reaction. After enzymatic removal of all N-glycans, the protonated molecular masses obtained were 49,399, 49,380 and 49,391 Da for the alpha-form, the beta-form and the unseparated natural mixture, respectively. These values are in good agreement (+0.026% for the alpha-form, -0.012% for the beta-form and +0.010% for the unseparated mixture) with the calculated molecular mass based on the SwissProt data. The determined molecular masses after reduction/alkylation and de-N-glycosylation of the alpha-and beta-forms are almost equal, indicating that no major differences exist between the three preparations on the amino acid level.

Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides.

Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid-phase isolation steps.

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration.

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.