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Under the auspices of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC/ISTH) a secondary plasma coagulation standard has been made available to manufacturers with the objective of improving the harmonization of labeled analytes in commercial plasma calibrants. The SSC/ISTH Secondary Coagulation Standard Lot no. 3 (SSC Lot no. 3) consists of vials each containing 1 mL of pooled normal human plasma, lyophilized, and has been assigned values for 19 coagulation-related analytes by assay relative to the relevant World Health Organisation International Standards (WHO IS) [1]. The present report describes the stability of SSC Lot no. 3 based on studies of accelerated degradation at elevated temperatures and ‘real-time’ storage at the bulk storage temperature of −20 °C which have been undertaken over a 6-year period in order to support the continued validity of the assigned values as despatch commenced in July 2006.

The standard (SSC Lot no. 3) was prepared from a plasma pool comprising 86 donations from normal, healthy donors, collected by plasmapheresis, each tested and found negative for viral markers for hepatitis B virus, hepatitis C virus and human immune-deficiency virus 1 and 2. The pooled plasma was filled in 1mL aliquots into 55 000 rubber-sealed, screw-capped glass vials and lyophilized. Mean residual moisture after freeze-drying was 0.101%. Four analytes [factors (F)V, VII, VIII and XI] underwent testing in two different laboratories (NIBSC, Potters Bar, UK and Royal Hallamshire Hospital, RHH, Sheffield, UK). FV and VII were estimated using thromboplastin-based one-stage clotting methods and FXI was estimated using activated partial thromboplastin time (APTT)-based one-stage clotting methods; FVIII was measured using the two-stage clotting method (RHH) and the chromogenic method (Chromogenix Coatest SP4 kit) (NIBSC). Stability was assessed through an accelerated degradation study which involved storage of vials of SSC Lot no. 3 at elevated temperatures (+4, +20, +37, +45 °C) since December 2003 with periodic sampling and residual potency estimation relative to vials stored at the reference temperature (−20 °C) after 2.2, 4.2 and 6.2 years storage. Each testing time-point involved four independent potency estimates for each analyte by each laboratory using different vials in each assay. Predictions of degradation rate were calculated according to the Arrhenius equation (ACDWin software version 8.18, P K Phillips) based on the assumption that degradation results from a uni-molecular decay process where the degradation at higher temperatures only differs from degradation at lower temperatures in terms of rate [2].

Real-time stability was assessed by the comparison of vials of SSC Lot no. 3 stored at −70 °C relative to vials stored at −20 °C after 6.2 years (eight independent estimates for each analyte).

Results from the two laboratories for samples stored at elevated temperatures agreed very closely in the estimates of residual potency for all four factors. Combined mean relative residual potency for vials stored at +4 °C for 6.2 years exceeded 90% for FV, FVII and FXI with the greatest loss observed for FVIII with 83% residual potency (Table 1a). For the higher storage temperatures (20, 37, 45 °C) the greatest loss was consistently found for FV and FVIII with a combined residual potency of 61% and 57%, respectively, after 6.2 years at 20 °C and 6% and 17%, respectively, after 4.2 years at 37 °C. All data points for the residual relative potencies fitted the Arrhenius model extremely well with good agreement between the observed and predicted loss. The predicted mean percent loss per year for vials stored at temperatures ranging from −20 to +37 °C is given in Table 1b. All four factors were associated with a mean predicted loss of < 0.1% per year at −20 °C. Predicted losses for storage at all of the higher temperatures confirmed that FV and FVIII were less stable than FVII and FXI. An estimate of the precision of the predictions can be obtained from the upper 95% CI of loss as given in Table 1b and, in all cases, the proximity of the upper limit with the mean value is an indication of a robust prediction. As a check on the validity of the predictions, the measured residual activity after 6.2 years of storage at +20 °C has been compared with the expected residual activity after 6 years calculated using the mean predicted loss in Table 1b. The results for the calculated and measured residual activities, respectively, were extremely close: FV 61.7% vs. 60.5%; FVII 85.1% vs. 85.5%; FVIII 57.0% vs. 57.0%; FXI 85.6% vs. 87.0%.

Table 1.   Accelerated degradation study for coagulation factors (F)V, VII, VIII and XI in Scientific and Standardization Committee (SSC) Lot no. 3. (a) Mean residual relative potencies after storage for 2.2, 4.2 and 6.2 years as a percent relative to vials stored at −20 °C. (b) Predicted mean percent loss per year for vials stored at −20, +4, +20 and +37 °C based on combined data
Storage temperature (°C)Factor V (%)Factor VII (%)Factor VIII (%)Factor XI (%)
2.2 years4.2 years6.2 years2.2 years4.2 years6.2 years2.2 years4.2 years6.2 years2.2 years4.2 years6.2 years
(a)
+495 (4.1)93 (6.8)90 (3.9)97 (3.8)102 (5.9)96 (1.9)98 (9.1)95 (10.4)83 (3.4)99 (3.7)101 (14.3)98 (1.7)
+2079 (2.9)68 (2.8)61 (2.0)94 (4.7)94 (5.4)86 (2.0)81 (7.9)68 (8.3)57 (1.9)93 (7.0)90 (12.4)87 (3.1)
+3724 (3.3)6 (5.8)70 (3.3)54 (4.9)34 (8.4)17 (2.7)57 (5.3)40 (11.6)
+457 (2.5)37 (5.7)7 (2.0)24 (5.0)
Storage temperature (°C)Factor VFactor VIIFactor VIIIFactor XI
  1. (a) Results are the combined mean values from four individual estimates each from NIBSC and the Royal Hallamshire Hospital with standard deviations (SD) in brackets; †Not tested owing to insolubility of lyophilized material. (b) Figures in brackets represent the upper 95% confidence limit (CI) of predicted loss (% per year).

(b)
−200.034 (0.056)0.015 (0.039)0.098 (0.207)0.004 (0.012)
+41.08 (1.41)0.40 (0.71)1.76 (2.59)0.24 (0.47)
+207.74 (8.62)2.66 (3.53)8.95 (10.48)2.55 (3.72)
+3741.60 (46.69)15.10 (20.33)36.65 (45.19)21.79 (29.78)

The real-time comparison of vials stored at −70 °C for 6.2 years with vials stored at the bulk storage temperature of −20 °C provided an objective assessment of the stability of the standard. Estimates from the two laboratories agreed closely and the combined mean relative estimates for all four coagulation factors (−70 as % of −20 °C) were 96% for FV, 99% for FVII, 96% for FVIII and 101% for FXI.

The analytes tested in the present study were chosen to represent examples of both stable (FVII, FXI) and labile (FV, FVIII) coagulation factors from the list of assigned values for SSC Lot no. 3. Assessment of stability was required not only to demonstrate that the preparation remained fit for purpose but also to support the assignment of an expiry date. Stability testing focussed on the accelerated degradation approach which has been applied to lyophilized WHO IS for several decades [2]. This model fitted the residual relative potency data extremely well and allowed the inclusion of the accumulated results from all three testing time-points (2.2, 4.2 and 6.2 years). The reliability of the predictions of mean percent loss per year was supported by the proximity of the upper 95% CI of loss for all factors and the excellent agreement between the real-time observed loss (at +20 °C) with the calculated loss based on the predicted degradation rate. The mean predicted loss for storage at −20 °C was < 0.1% per year for all four factors and this indicates that the preparation is extremely stable at the bulk storage temperature (−20 °C). This level of stability is similar to that previously reported for lyophilized WHO IS which are presented in sealed glass ampoules [3,4]. The results from the present study demonstrate that rubber-sealed vials can produce equally stable preparations subject to suitable formulation and other conditions including residual moisture. Excellent stability of the four factors during −20 °C storage is also supported by the results of the real-time comparison with −70 °C vials after 6.2 years storage.

Calculation of the expiry date for SSC Lot no. 3 was based on the predicted stability of the most labile factor (FV) identified from the accelerated degradation study after 2.2 years storage at elevated temperatures. Here FV was associated with a mean predicted loss of 0.18% per year at −20 °C with an upper 95% CI of 0.46%. The shelf-life was calculated as the period during which the residual potency remained above 95% based on the predicted upper 95% CI of loss and was equivalent to a maximum of 11 years. The actual expiry date of ‘end 2014’ was agreed in 2006 and fell within this period. Subsequently the inclusion of data from all testing time-points (2.2, 4.2 and 6.2 years) has generated a more robust prediction of loss and FVIII appears to be the most labile of the four factors. The upper 95% confidence limit of loss of 0.207% for FVIII is consistent with a shelf-life exceeding 20 years and hints that the earlier assignment of shelf-life under-estimated the stability of the preparation.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interest
  4. References

The support of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis is gratefully acknowledged.

Disclosure of Conflict of Interest

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interest
  4. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interest
  4. References