Frozen and freeze-dried solvent/detergent treated plasma: Two different pharmaceutical formulations with comparable quality.

BACKGROUND: OctaplasLG is a frozen solvent/detergent-treated plasma product used for treating complex coagulation factor deficiencies or as substitution therapy in emergency situations where specific factor concentrates are not available. A new freeze-dried (also known as lyophilized) form of OctaplasLG, referred as OctaplasLG Lyo (Octapharma AG, Switzerland) offers rapid reconstitution and more flexible storage conditions, improving logistics and utilization. This study compared the biochemical quality of OctaplasLG Lyo with OctaplasLG and single-donor fresh frozen plasma units. STUDY DESIGN AND METHODS: Three batches of OctaplasLG Lyo, manufactured for production process qualification, and 12 batches of OctaplasLG were provided by Octapharma AB (Sweden). Twelve units of fresh frozen plasma were collected by the local FDA-licensed blood provider. All plasma samples were assessed for global coagulation parameters, coagulation factors and protease inhibitors, activation markers of coagulation and fibrinolysis, and important plasma proteins. Quality control assays were conducted in accordance with European Pharmacopeia requirements. RESULTS: Frozen and freeze-dried OctaplasLG demonstrated comparable quality profiles upon thawing or reconstitution. All coagulation factor and protease inhibitor activity parameters were in line with levels mandated by the European Pharmacopeia. Fresh frozen plasma units showed comparable coagulation factor activities, with higher protein S and plasmin inhibitor levels than the OctaplasLG products. Fresh frozen plasma parameters showed high lot-to-lot variations. DISCUSSION: The two pharmaceutical forms of OctaplasLG (frozen and freeze-dried) have comparable biochemical quality. Key features of OctaplasLG Lyo are rapid reconstitution time and storage flexibility, which may improve logistics and utilization, and have particular advantages in emergency situations and pre-hospital settings.

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.

Five-day stability of thawed plasma: solvent/detergent-treated plasma comparable with fresh-frozen plasma and plasma frozen within 24 hours.

BACKGROUND: Plasma stored refrigerated for up to 5 days after thawing is common practice in many US hospitals. Therefore, clotting factor activities in fresh-frozen plasma (FFP), plasma frozen within 24 hours (PF24), and solvent/detergent-treated plasma (SDP), thawed and stored at 1 to 6°C for up to 5 days, were investigated. STUDY DESIGN AND METHODS: Five A, B, O, and AB units of FFP, PF24, and SDP were thawed and maintained for 5 days at 1 to 6°C. The activity of factor (F)V, FVII, FVIII, protein S (PS), and ADAMTS13 was determined in each unit at baseline and every 24 hours thereafter for 5 days. RESULTS: After thaw, mean values of the variables tested were within the normal range in all three plasma products although, in SDP, FVIII activity was significantly lower (p = 0.0039). After 5 days of storage all factors significantly declined except for ADAMTS13 activity, which was stable. Mean FVIII and ADAMTS13 activity was comparable in all three plasma products and within the normal range, mean FV activity was significantly lower in FFP and PF24 (p<0.0001) compared to SDP, and mean FVII activity was significantly lower in PF24 (p<0.03) than in FFP or SDP. Mean PS activity was below the normal range in all three plasma products with the lowest values in SDP (p = 0.0001). CONCLUSION: Over 5 days of refrigerated storage the changes in the measured coagulation factors in FFP, PF24, and SDP are comparable. Clinical follow-up is needed to assess whether slightly lower PS levels in SDP are clinically important.

Universal pooled plasma (Uniplas(®)) does not induce complement-mediated hemolysis of human red blood cells in vitro.

BACKGROUND: Pooling of plasma of different blood groups before large scale manufacturing of Uniplas(®) results in the formation of low levels of soluble immune complexes (CIC). The aim of this study was to investigate the level and removal of CIC during Uniplas(®) manufacturing. In addition, an in vitro hemolysis assay should be developed and investigate if Uniplas(®) does induce complement-mediated hemolysis of human red blood cells (RBC). MATERIALS AND METHODS: In-process samples from Uniplas(®) (universal plasma) and Octaplas(LG)(®) (blood group specific plasma) routine manufacturing batches were tested on CIC using commercially available ELISA test kits. In addition, CIC was produced by admixing heat-aggregated immunoglobulins or monoclonal anti-A/anti-B antibodies to plasma and removal of CIC was followed in studies of the Uniplas(®) manufacturing process under down-scale conditions. The extent of RBC lysis was investigated in plasma samples using the in-house hemolysis assay. RESULTS: Levels of CIC in Uniplas(®) are within the normal ranges for plasma and comparable to that found in Octaplas(LG)(®). Down-scale experiments showed that both IgG/IgM-CIC levels are significantly removed on average by 40-50% during Uniplas(®) manufacturing. Uniplas(®) does not induce hemolysis of RBCs in vitro. Hemolysis occurs only after spiking with high titers of anti-A/anti-B antibodies and depends on the antibody specificity (i.e. titer) in the plasma sample. CONCLUSION: The results of this study confirm the safety of Uniplas(®) regarding transfusion to patients of all ABO blood groups.

Clotting factor activity in thawed Octaplas® LG during storage at 2-6°C for 6 days from a quality assurance point of view.

INTRODUCTION: Octaplas® LG is a second-generation solvent/detergent-treated plasma that offers an additional safety benefit by prion elimination. The stability of clotting factors of the new S/D plasma after thawing has not been investigated yet. This study intended to measure the time course of fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI through storage at 2-6°C over 6 days. MATERIALS AND METHODS: We investigated 20 plasma bags (five bags per blood group) and measured fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI immediately after thawing and after 2, 4, 6, 24, 48, 72, 96, 120 and 144 h storage at 2-6°C. Five separate plasma bags were thawed and stored at 2-6°C for microbiological assessment. After 6 days samples were drawn for blood cultures that were incubated for six more days. RESULTS: After 6 days FII, FIX and PC showed no significant changes. FV (-16%, p<0.001), FVII (-19%, p<0.001), FVIII (-19%, p<0.001), FXI (-13%, p<0.0001) and fPS (-4%, p<0.0007) decreased significantly. PI levels were stable at 56%. The microbiological investigation showed no bacterial contamination. CONCLUSIONS: In Octaplas® LG plasma clotting factors decreased slightly through storage of 6 days. PI levels were remarkably higher and stable over time in the new Octaplas® LG. Stability of stored Octaplas® LG was limited by the decrease of FVIII to 53%, which may warrant storage up to 24h from a quality assurance point of view. This could result in reduced plasma wastage and costs for healthcare givers.

Prion safety of transfusion plasma and plasma-derivatives typically used for prophylactic treatment.

Reports about transfusion-related transmissions of variant Creutzfeldt-Jakob disease have urged the need for more information regarding the risk for prion contaminated units in the blood supply and the safety of transfusion plasma and biopharmaceuticals derived from this precious raw material. According to a possible epidemiological model, the risk in many European countries is the same or lower than that of human immunodeficiency virus. Comprehensive investigations have shown that the prion safety margin of both single-donor and pooled solvent/detergent treated transfusion plasma is high. Furthermore, prophylactic treatment using plasma-derivatives poses a very low risk in terms of prion disease despite extensive lifetime exposure.

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.

A biochemical comparison of a pharmaceutically licensed coagulation active plasma (Octaplas) with a universally applicable development product (Uniplas) and single-donor FFPs subjected to methylene-blue dye and white-light treatment.

The strive for more standardised and highly efficacious products is one of the important mainstays in modern haemotherapy. Coagulation active plasma for transfusion is the product of choice when treating hereditary or acquired isolated or complex coagulopathies, when no specific concentrate is available. The aim of this study was to perform an extensive biochemical comparison of the pharmaceutically licensed coagulation active plasma named Octaplas with an identical, but universally applicable, development product (Uniplas, working title) and single-donor fresh-frozen plasma (FFP) units subjected to a medical device treatment using a combination of methylene-blue dye and subsequent white-light exposure (MB plasma). Our study showed that there are differences in the biochemical characteristics between Octaplas and MB plasma, while Uniplas revealed the same quality as Octaplas. The variability of selected plasma proteins in the 20 individual MB plasma units tested was high compared to Octaplas/Uniplas. Beyond the reported decreased levels of protein S and plasmin inhibitor found in Octaplas/Uniplas, and the significant loss of fully functional fibrinogen in MB plasma and its impact on selected global coagulation parameters, the latter product additionally revealed several coagulation factor activities outside the ranges given for normal single-donor FFP. It is important for plasma prescribers to be aware of the major inherent differences between Octaplas and MB plasma.

Stability of solvent/detergent-treated plasma and single-donor fresh-frozen plasma during 48 h after thawing.

The aim of this study was to compare the quality of solvent/detergent (SD) treated plasma, Octaplas, and single-donor fresh-frozen plasma (FFP) units during 48-h storage after thawing. Octaplas bags of different blood groups and individual FFP units were thawed and stored at either +4 degrees C or at room temperature (RT) for 48 h. Samples drawn during the observation period were investigated on various coagulation factor and protease inhibitor activities using standard coagulation and chromogenic assays. The generation of FVIIa was followed as a marker of coagulation factor activation. All investigated coagulation factors and protease inhibitors were stable for at least 8h during storage of Octaplas at +4 degrees C. FVIII levels started to decline earlier in FFP than in Octaplas at both storage temperatures. Stored Octaplas OD660 values were more stable during the storage period than FFP OD660 values, whereas VWF multimeric patterns were comparably stable in both types of plasma. In conclusion, this stability study has demonstrated that thawed Octaplas maintains its high quality, even with a time safety margin, for 8 h at +4 degrees C and for 6 h at RT. In general, there was more variability in coagulation factor levels among different FFP units compared with different Octaplas batches.

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.

Separation of active and inactive forms of human antithrombin by heparin affinity chromatography.

During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human alpha-antithrombin, human beta-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 degrees C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the alpha-latent and beta-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods.

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.