Original Papers 01991 S. Karger AG,Basel M)42-9007/9y0603-0141$2.75/0
Vox Sang 1991;60:141-147
Large-Scale Preparation of a Highly Purified Solvent-Detergent Treated Factor VIII Concentrate Robert Myersa, Milan Wickerhausera,Leigh Charamellaa,Louise Simon ",William Nummy a, Teresa Brodniewicz-Proba "Blood Derivatives Section of the Michigan Department of Public Health, Lansing, Mich., USA bBlood Fractionation Center 'Armand-Frappier', Laval, Quebec, Canada
Abstract. Large-scale adaptation of a recently reported glycine precipitation method for the production of factor VIII (FVIII) concentrate is described. Scaling up of the method required some modification including the addition of aluminum hydroxide to the glycine buffer to reduce the level of contaminating proteins in the final preparation and the use of centrifugation to replace filtration by glass beads. Furthermore, the resultant product was virus inactivated by incorporation of the organic solvent and detergent technique. At industrial level, the modified method gave a good recovery of FVIII activity (230 IU/1 plasma) with high purity (4IU/mg protein). The final product, after virus inactivation and lyophilization, yielded 185 IU of FVIII activity per liter of starting plasma and was considered to be suitable for clinical evaluation.
Introduction While the industrial trend toward purer preparations of factor VIII concentrates (FVIII) for clinical use continues, shortages of the preparation have occurred and the benefit of the purer products has come into question, given their high price [l-31. As of now, clinically relevant immunosuppression has not been clearly demonstrated for intermediate or high-purity FVIII preparations [l, 4,5]. While this possibility is being further explored there will be a continued need for cost-efficient products of high recovery well into the future. Procedures employing precipitation of impurities by glycine, either solid or in solution [&lo], have been known for some time and these methods have been reported to yield a high recovery of FVIII activity (FVII1:C) and to be of acceptable purity. Recently, a small-scale procedure for the preparation of FVIII was described [ll], based on modifications of a previously reported procedure using glycine and sodium chloride (NaCl) as precipitants [8]. This procedure gave a good recovery of FVII1:C (280 IU/l), at a specif-
ic activity of 10 IU/mg protein and resulted in a stable preparation both in liquid state and during severe dry-heat (8OOC) treatment [ll]. However, the procedure was not scaled up to a clinically useful production size and uncertainty remained in its suitability for use with industrial scale equipment. Additionally, the careful batch-thawing and cryoprecipitate collection described by Brodniewicz-Proba et al. [ll]could not be easily duplicated on a large scale, and it was not known whether cryoprecipitate collected on an industrial scale could be processed to FVIII in a similar manner. Finally, although this preparation was shown to be suitable for the clinically promising [Elsevere dry-heat treatment, its suitability for virus inactivation by more widely accepted methods such as pasteurization [lo] or solventdetergent ( S K I ) treatment [13] was not evaluated. The objectives of the present study were to scale-up the modified glycine precipitation procedure for the preparation of FVIII and to incorporate the S / D virus inactivation technique. The work described here allowed large-scale preparation of an economical, virus-inactivated FVIII concentrate.
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Materials and Methods The starting material for the preparation of FVIII was fresh-frozen plasma from volunteer donors and was obtained from the three Michigan Regions of the American Red Cross. The plasma was separated from whole blood, rapidly frozen within 15 h of collection and stored at or below -30°C until use.
Buffers Tris Buffer. 0.02 M tris-(hydroxymethy1)aminomethaneadjusted to pH 7.0 with HCI. Glycine Buffer. 2.8 M glycine containing 0.3 M NaCl and 0.025 M Tris adjusted to pH 6.8 with HCI. Redissolving Bufer. 0.02 M trisodium citrate containing 0.005 M calcium chloride, 0.04 M NaCl and 1% sucrose, adjusted to pH 7.1 with HCI. Wash Buffer. 1.0 M glycine containing 15% NaCI, 0.02 M trisodium citrate and 0.005 M calcium chloride.
Fig. 1. Isolation of FVIII concentrate.
Final Buffer. 0.02 M sodium citrate containing 0.04 M glycine, 0.06 M NaCI, 0.005 M calcium chloride and 1.5% sucrose, adjusted to pH 7.1 with HCI. Assays FVI1I:Cwas determined by a one-stage automated activated partial thromboplastin time technique on a Lancer Coagulyzer I1 (Sherwood Medical, St. Louis, Mo., USA), using an intermediate-purity FVIII reference standard (Mega-I, 10.2 IU/ml, USFDA) and congenitally FVIII-deficientplasma (George King, Overland Park, Kans., USA) as substrate. FVIII-related antigen (vWf Ag), albumin and fibronectin were measured by rocket immunoelectrophoresisaccording to Laurel1 [14]. Total protein was determind by the biuret method [15], using human albumin as a protein standard. Immunoglobulin gamma (IgG) was measured by radial immunodiffusion. Clottable fibrinogen was determined by a modification of the Clauss method [16]. Tri(n-buty1)phosphate (TNBP) and Ween 80 (T80) were quantitated by gas-chromatographic and spectrophotometric procedures respectively as recommended by Horowitz et al. [13]. Sodium was determined by flame photometry. The absence of any thrombin was ascertained by incubating samples of FVIII concentrate with a 1% solution of fibrinogen in plastic tubes at room temperature for 24 h and observing no sign of clot from intrinsic fibrinogen [17]. Reconstitution time was measured by adding sterile water for injection and gently shaking the vial until no particles were visible. The rabbit pyrogen test was conducted according to prescribed methods [18]. The dose was 1.0 mi (40-50 IU) of reconsituted FVIII concentrate per kilogram of body weight and the results were reported as the sum of the temperature increase of the three rabbits in each test. Preparation Procedure Isolation of FVII1:C. Individual units of plasma sufficient for pools of 620-1,480 liters were removed from -30°C storage and allowed to equilibrate at -10°C overnight. Units of plasma were extruded from satellite bags, crushed, pooled and thawed by the contiuous-thaw method [19]. Cryoprecipitate was harvested in Sharpies AS-16 centrifuges (Pennwalt Corp. Warminster, Pa., USA) in the continuous-
flow mode with supernatant temperature controlled at 0-+2"C. The precipitate was then redissolved in Tris buffer in the proportion of 30 mM of starting plasma, at a temperature of 28-30°C. Glycine buffer, enriched with a 2% aluminum hydroxide suspension (Rehsorptar, Armour), was then added to final concentrations of about 2.0 M glycine and 6.7 ml A1(OH)3per liter of starting plasma. After brief mixing and standing periods, this suspension was centrifuged in a Westfalia centrifuge (Centrico, Inc., Engelwood, N.J., USA) in the continuous-flow mode with supernatant temperature controlled at 28-34°C. Pilot and production scale batches utilized the Westfalia models KO2006 and KDD605 centrifuges, respectively. Granular NaCl was then added to the supernatant to a final concentration of 10% under gentle mixing at a temperature of 28-30°C. After a brief standing period, this suspension was centrifuged in Sharples AS-16 centrifugeswith supernatant temperature controlled at 25-32°C. The NaCl precipitate thus obtained was rapidly frozen and stored at -70°C. The flow diagram for this portion of the process is depicted in figure 1.
viral Inactivation and SID Removal. For viral inactivation, from one to four individual batches of frozen NaCl precipitate were removed from storage and added to redissolving buffer in the proportion of 4.0 ml buffer per liter of starting plasma. The precipitate was dissolved with vibromixing and brought to a temperature of 28-30°C. To remove particulates that may affect the efficacy of viral inactivation, this solution was clarified first by batchwise centrifugation in a Sorvall RC2B centrifuge (DuPont Co., Wilmington, Del., USA) and then by filtration through a 0.45ym filter. After pH adjustement to 6.95-7.0, TNBP and T80 (both from Fisher Scientific) were added to final concentrations of 0.3 and LO%, respectively, followed by incubation for 6 h at 28-30°C under gentle stirring. At the conclusion of incubation, the volume was doubled by the addition of redissolvingbuffer. Precipitation was accomplishedby addition of granular glycine and NaCl under vibromixing to achieve final concentrations of 1.0 M and 15%. respectively. After a brief standing
Preparation of Virus-Inactivated Factor VIII Concentrate
Fig. 2. Flow diagram for the virus inactivation, S / D removal and final FVIII preparation.
period at 28-30°C, the suspension was centrifuged batchwise in a Sorvall RC3B (DuPont Co. Wilmington, Del., USA) for 30 min at 6,000 g. The supernatant was then decanted and the precipitate was thoroughly rinsed with the wash buffer. After dissolving the precipitate in the redissolving buffer at a proportion of 8.0 mM of starting plasma, precipitation was repeated. The second precipitate was redissolved in the final buffer at a proportion of 4.0 mM of plasma. The solution was then diafiltered against lvol of the final buffer in a Benchmark@ rotary ultrafiltration unit (Membrex, Inc., Garfield, N.J.,USA), supplied with a membrane constructed of surface-modified polyacrylonitrile and of 100,OOO molecular weight cut-off. After sterile filtration through a 0.22-pn filter, the final preparation of FVIII was dispensed into 50-ml vials in IO-ml volumes and lyophilized in a Hull Model 24FS40C Lyophilizer (Hull C o p , Hatboro, Pa., USA) at a product temperature below -30°C during primary drying and +25"C during secondary drying. The flow diagram for the virus inactivation, S / D removal and final FVIII preparation is depicted in figure 2.
Results Scale- Up Studies Preliminary pilot-scale experiments were conducted to examine the feasibility of the glycine procedure [ll] with the cryoprecipitate collected by large-scale techniques, in our case by the continuous-thaw method. The results were disappointing, with a mean FVII1:C recovery of 220 IUA plasma and specific activity of 1.9 IU/mg protein (n = 5 ) . The low specific activity prompted an evaluation of the
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Fig.3. Recovery of FVI1I:C and specific activity in the sodium chloride precipitate at different levels of aluminum hydroxide in the combined aluminum hydroxide/glycine step. Plotted values represent the mean of 3 experiments.
addition of an Al(OH)3 adsorption step. Various levels of Al(OH)3 were evaluated either as a separate step before glycine precipitation or combined into a single step (data not shown). A greater improvement in specific activity was obtained in the combined step, so a series of experiments was undertaken to determine the optimal A1(OH)3 concentration for the combined adsorptiodprecipitation step. The results of this evaluation are illustrated in figure3. The optimal level of Al(OH)3was found to be about 6.7 ml of a 2% suspension per liter of starting plasma. Below this level, specific activity diminished while above this level, FVII1:C recovery rapidly deteriorated with no significant improvement in specific activity. The method for separation of the FVII1:C-rich supernatant from the combined Al(0H)dglycine suspension by filtering through glass beads as described for the small-scale procedure [ll]was not suitable for large-scale application. Based on the amount of glass beads described, the weight of glass beads required for 1,OOO liters starting plasma was estimated to 440 kg. In addition, preliminary experimentation revealed filtration to be slow and that a significant amount of FVIII-rich liquid was lost in the column and in the loosely formed precipitate. As an alternative, two types of continuous-flow centrifuges were evaluated. The Sharples centrifuge created a problem as it caused a 65% loss in the FVII1:C activity presumably due to heavy foaming of the supernatant. On the other hand, continuous-flow centrifugation with the Westfalia centrifuges proved most use-
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Table 1. Pilot (150-200 liters plasma) and production scale (500-620 liters plasma) purification of FVIII
Stage
Cryoprecipitate Glycine/A1(OH)3supernatant NaCl precipitate
FVIII recovery IUA plasma
Total protein recovery mgh plasma
FVIII specific activity IU/mg protein
pilot
production
pilot
production
pilot
production
452k45 307f31 254k39
409 f68 288f32 230f30
1460f181 794k116 67k15
1375f177 808f47 61k11
0.31+0.02 0.37f0.10 4.0fl.l
0.30f0.05 0.38fO.08 3.9f0.7
Results are means f l SD; n = 10 for pilot and n = 10 for production scale.
ful and resulted in minimal loss of VII1:C activity. The supernatant at this step could be separated in production lots from 620 liters starting plasma in 30 min, with a flow rate of about 200 litersh. Mean recovery of FVII1:C activity and specific activity for ten pilot and ten production scale batches are shown in table 1. Overall, FVII1:C recovery was identical (56%) for pilot and production scale batches, based on the starting activity in the cryoprecipitate. Total protein recovery across the two steps was also similar, yielding 4.6 vs. 4.4% for pilot and production scale batches, respectively, based on the total protein in the cryoprecipitate. Activity of FVII1:C in the NaCl precipitate fraction was found to be stable for several months when rapidly frozen and stored at -70°C. The NaCl precipitate averaged 0.70 g/1 of starting plasma. Because of its stability and small mass, this fraction provided a convenient stopping point in the production process and made possible the viral inactivation and final preparation of large batches without the need for increasing the size of the equipment used to obtain the NaCl precipitate from plasma.
SID Reagent Removal Studies Primary focus on these experiments was to develop a reliable method for removal of TNBP and T80. Precipitation was chosen as the method of removal and experiments were designed to determine ways to optimize recovery of FVII1:C while at the same time reducing TNBP and T80 to clinically acceptable levels. In preliminary experiments, it was established that a single precipitation with glycine and NaCl was insufficient for removal of TNBP and T80. Under the conditions of optimal FVII1:C recovery, a single precipitation reduced TNBP and T80 to 350parts per million (ppm) and 1,300ppm, respectively. For further removal of the re-
Fig.4. Effect of glycine and sodium chloride concentrations on recovery of FVI1I:C and residual levels of TNBP and T80 after two successive precipitations. Plotted values represent the mean of 5 experiments.
agents, two successive precipitation steps using various final concentrations of NaCl and glycine were evaluated. The results of these experiments are shown in figure 4. As can be seen, optimal recovery of FVII1:C and maximum reduction of TNBP and T80 were achieved by two successive precipitations at 1.0 M glycine and 15% NaCl. Under these optimized conditions, the levels of TNBP were reduced to 13-22 ppm and those of T80 to 15-35 ppm. Ultrafiltration was then evaluated to further remove TNBP. It was found that TNBP was consistently reduced to a level of 2ppm or less after diafiltration against a single volume of final buffer.
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Preparation of Virus-Inactivated Factor VIII Concentrate
Table 2. Recovery and specific activity of FVIII at various steps of the viral inactivation process Stage
Redissolved, clarified NaCl precipitate Postviral inactivation precipitate 1 Postviral inactivation precipitate 2 Diafiltrated solution Sterile bulk product Final lyophilized product
FV1II:C recovery IUA plasma
Total protein recovery mgll plasma
Specific activity IU/mg protein
239f28
63f12
3.9 f0.8
217f32
57f12
3.9f0.8
218f25 212f20 197f20
52f11 51f11 47fll
4.4f0.9 4.3 f0.9 4.4f0.9
185f17
46f8
4.1f0.6
Results are meanfSD for 9 lots of FVIII.
Several large-scale batches of FVIII were then prepared. Recovery of FVIII:C, total protein and specific activity are summarized in table 2. The relative amounts of FVII1:C activity and protein recovered across each step were similar except for the second precipitation which appeared to result in slight purification. Overall, the FVII1:C recovery for the viral inactivation and S / D reagent removal steps was 89%. Handling losses during sterile filtration were 7% and FVII1:C loss across lyophilization was 6%. Characteristics of the final, virus-inactivated, lyophilized FVIII concentrate for nine large-scale research and clinical trial batches are shown in table 3. The reconstitution time of the product was about 3min with a specific activity of 4 IU/mg of protein and the stability of the reconstituted FVIII (24 h at room temperature) was approximately 90%. Clottable fibrinogen was about 55% of the total protein. Fibronectin, albumin and immunoglobulin gamma were present in much lower concentrations. These individual protein assays accounted for approximately 62% of the total protein present and no attempts were made to identify the remaining proteins. TNBP and T80 were present only in trace amounts of about 1 and 10 ppm, respectively. Processing time at the large-scale required about 4 h to obtain NaCl precipitate from cryoprecipitate, 7 h for redissolving and viral inactivation and 9 additional hours to obtain the sterile filtered final bulk FVIII concentrate. Although steps during most of this 20-hour time period were
Table 3. Characteristics of large-scale produced, S/D-treated FVIII Reconstitution time, s Appearance FVI1I:C IUlml 24-hour reconstituted stability (24 h at 22"C), YO Protein, mg/ml Specific activity, IU/mg protein vWf:Ag, Ulml Clottable fibrinogen, mg/ml Fibronectin, mg/ml Thrombin test (24 h at room temperature) PH Albumin, mg/ml IgG, mg/ml m p PPm n e e n 80, ppm Sodium, mEqA Rabbit pyrogen test, "C
200f63 clear, colorless 44.6f4.4 89f11 11.4f2.4 4.0f0.5 90f29 6.3f2.1 0.69f0.14 negative 6.8fO.09 0.027f0.0068 0.085f0.060 0.97f0.76 11.7f5.8 138f9.3 0.2 f0.2
Results are mean +1SD for 9 lots of FVIII.
conducted at 30°C, rabbit pyrogen test results were consistently acceptable, ranging from no increase to a sum total increase of 0.5 "C in body temperature.
Discussion Much progress in the development of virus-safe, pure FVIII preparations was accomplished in recent years, but it seems that there is still a need for preparation of economical FVIII concentrates [l-31. A recently reported procedure consisting of the preparation of FVIII by modified glycine precipitation, followed by NaCl precipitation resulted in an FVIII preparation of high purity and good recovery [ll]. Although the method was promising, certain aspects of the small-scale production technique were not easily adaptable to large-scale preparation, and a widely accepted virus-inactivation technique had not been incorporated into the process. For these reasons, a study was undertaken to modify the procedure and incorporate a S / D virus inactivation step. Early experiments revealed that the recovery and purity of FVIII obtained by the modified glycine procedure [ll] could not be duplicated using cryoprecipitate collected by large-scale methods. In retrospect, this is not surprising. It is well known that both the recovery of FVII1:C and the level of contaminating proteins in cryoprecipitate may be significantly influenced by the methods for thawing of plasma and collection of cryoprecipitate [20, 211. Subsequent
146
recovery and purification of FVIII may also be affected by the method of thawing and cryoprecipitate collection [19]. The addition of A1(OH)3 to the glycine buffer more than doubled the specific activity of the NaCl precipitate with no additional loss of FVII1:C. The use of an A1(OH)3 adsorption step is not new for conventional techniques for isolation and purification of FVIII [lo, 22-26] but to our knowledge the present study is the first report of combining A1(OH)3 adsorption with the glycine precipitation into a convenient, single step. The diminished FVII1:C recovery at higher concentrations of Al(OHX probably results from a specific adsorption of FVII1:C [25,27]. The successful application of commonly used centrifugation equipment enhances the feasibility of the procedure for large-scale production of FVIII. Its use in the continuous flow mode allows the processing of increased batch sizes without the need to acquire larger equipment and permits this procedure to be easily incorporated into conventional plasma fractionation facilities. The S / D viral inactivation method was chosen because of its demonstrated virucidal capability and its relatively thorough clinical evaluation [2, 28, 291. The nonionic detergent T80 was chosen in lieu of sodium cholate because of the latter’s reported inactivation of FVII1:C during incubation [13]. The redissolved NaCl precipitate fraction was chosen for viral inactivation because of its relatively small volume. This fraction, redissolved as described in the present study, results in a volume of only 4.4 liters for the virus inactivation step from a starting plasma volume of 1,000 liters. This small volume facilitates the performance of virus inactivation and reduces the space and equipment requirements for segregated postviral inactivation-processing steps. The removal of S / D reagents by precipitation was chosen in part for its technical simplicity. Although the development of precipitation techniques required considerable experimentation, they proved to be effective in removing T80 to acceptable levels, overcoming the previously reported failure attributed to micelle formation [13]. Diafiltration was required to further reduce TNBP to levels safely below the acceptable upper limit pf 10ppm (New York Blood Center product specification). Interstingly, there was an over 90% reduction of TNBP with diafiltration against a single volume of the final buffer. This might be due to partial absorption of TNBP to the ultrafilatration membrane. With this S / D reagent removal method, a virus-safe FVIII concentrate is obtained with minimal losses in FVIII:C activity. The final lyophilized product is considered acceptable for clinical use in all respects and is now undergoing clinical evaluation.
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References 1 Brettler DB, Levine PH: Factor concentrates for treatment of hemophilia: Which one to choose? Blood 1989;73:2067-2073. 2 Pierce GC, Lusher JM, Brownstein AP, et al: The use of purified clotting factor concentrates in hemophilia. Influence of viral safety, cost and supply on therapy. JAMA 1989;261:3434-3438. 3 Aledort LM, Hilgartner M, Lipton RA, et al: Hemophilia - A treatment in crisis. N Engl J Med 1988;319:1017. 4 Teitel JM, Freedman JJ, Garvey MB, et al: Wo-year evaluation of clinical and laboratory variables of immune function in 117 hemophiliacs seropositive or seronegative for HIV-1.Am J Hematol 1989;32:262-272. 5 Brettler DB: Proposed protocol for the evaluation of the effect of high purity concentrates on the immune system of hemophilia patients. Thromb Haemost 1989;62:811-812. 6 Nilsson IM, Blomback M, Blomback B: Von Willebrand‘s disease in Sweden. Its pathogenesis and treatment. Acta Med Scand 1959;164:263-278, 7 Blomback B, Blomback M: Purification of human and bovine fibrinogen. Arkiv Kemi 1956/57;10:415-443. 8 Thorell L, Blomback B: Purification of the factorVIII complex. Thromb Res 1984;35:431450. 9 Winkelman L, Owen NE, Evans DR, et al: Severely heated therapeutic factor VIII concentrate of high specific activity. Vox Sang 1989$7 :97-103. 10 Heimburger N, Schwinn H, Gratz P, et al: Faktor VIII-Konzentrat, hochgereinigt und in Liisung erhitzt. Arzneimittelforschung 1981;31:619-622. 11 Brodniewicz-Proba T, Beauregard D: Modified glycine precipitation technique for the preparation of factor VIII concentrate of high purity and high stability. Vox Sang 1987;52:10-14. 12 Winkelman L, Feldman PA, Evans DR: Severe heat treatment of lyophilized coagulation factors; in Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Curr Stud Hematol Blood Ttansfus. Basel, Karger, 1989, No 56;pp 55-69. 13 Horowitz B, Wiebe ME, Lippin A, et al: Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-envelopedviruses by tri(n-buty1)phosphate detergent combinations. Transfusion 1985; 25 :5 16-522. 14 Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966;15:45-52. 15 Gornall AG, Bardavill CJ,David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949; 177:751-766. 16 Okuno T, Selenko V Plasma fibrinogen determination by automated thrombin time. Am J Med Tech 1972;38:196-201. 17 Middleton SM, Bennett IH, Smith JK: A therapeutic concentrate of coagulation factors 11, IX and X from citrated, factorVII1-depleted plasma. Vox Sang 1973;24:441456. 18 The United States Pharmacopeia, 21st rev. 1985, pp 1181-1182. 19 Foster PR, Dickson AJ, McQuillan TA, et al: Control of large-scale plasma thawing for recovery of cryoprecipitate factor VIII. Vox Sang 1982;42:180-189. 20 Mason EC, Pepper DS, Griffin B: Production of cryoprecipitate of intermediate purity in a closed thaw-siphon process. Thromb Haemost 1981$6543446. 21 Prowse CV, McGill A: Evaluation of the ‘Mason’ continuous-thaw siphon method for cryoprecipitate production. Vox Sang 1979; 37:235-243.
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Preparation of Virus-Inactivated Factor VIII Concentrate
22 Wagner RH, Richardson BA, Brinkhous KM: A study of the separation of fibrinogen and antihemophilic factor (AHF) in canine, porcine, and human plasma. Thromb Diath Haemorrh 1957;1:1-8. 23 Hershgold EJ, Pool JG, Pappenhagen AR: The potent antihemophilic globulin concentrate derived from a cold insoluble fraction of human plasma: characterization and further data on preparation and clinical trial. J Lab Clin Med 1966;67:23-32. 24 Newman J, Johnson AJ, Karpatkin MH,et al: Methods for the production of clinically effective intermediate and high-purity factor-VIII concentrates. Br J Haemat 1971;l-20. 25 Wickerhauser M, Mercer JE, Eckenrode Jw: Development of large-scale fractionation methods. VI. An improved method for preparation of antihemophilic factor. Vox Sang 1978;35:18-31. 26 Horowitz B, Lippin A, Chang MY, et al: Preparation of antihemophilic factor and fibronectin from human plasma cryoprecipitate. Transfusion 1984;24:357-362. 27 Seghatchian MJ, Kemball-Cook G, Barrowcliffe TW: Adsorption of factor VIII by aluminum hydroxide. Haemostasis 1979;8:106116.
28 Centers for Disease Control: Safety of therapeutic products used for hemophilia patients. MMWR 1988;37:441450. 29 Horowitz B: Investigations into the application of tri(n-buty1)phosphate/detergent mixtures to blood derivatives; in Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Curr Stud Hematol Blood Transfus. Basel, Karger, 1989, No 56; pp 83-86.
Received: May 14,1990 Revised manuscript received: September 20,1990 Accepted: October 10,1990 Robert C. Myers, DVM Section of Blood Derivatives Michigan Department of Public Health 3500 N. Logan Street PO Box 30035 Lansing, MI 48909 (USA)
Vox Sang 1991;60:141-147
Large-Scale Preparation of a Highly Purified Solvent-Detergent Treated Factor VIII Concentrate Robert Myersa, Milan Wickerhausera,Leigh Charamellaa,Louise Simon ",William Nummy a, Teresa Brodniewicz-Proba "Blood Derivatives Section of the Michigan Department of Public Health, Lansing, Mich., USA bBlood Fractionation Center 'Armand-Frappier', Laval, Quebec, Canada
Abstract. Large-scale adaptation of a recently reported glycine precipitation method for the production of factor VIII (FVIII) concentrate is described. Scaling up of the method required some modification including the addition of aluminum hydroxide to the glycine buffer to reduce the level of contaminating proteins in the final preparation and the use of centrifugation to replace filtration by glass beads. Furthermore, the resultant product was virus inactivated by incorporation of the organic solvent and detergent technique. At industrial level, the modified method gave a good recovery of FVIII activity (230 IU/1 plasma) with high purity (4IU/mg protein). The final product, after virus inactivation and lyophilization, yielded 185 IU of FVIII activity per liter of starting plasma and was considered to be suitable for clinical evaluation.
Introduction While the industrial trend toward purer preparations of factor VIII concentrates (FVIII) for clinical use continues, shortages of the preparation have occurred and the benefit of the purer products has come into question, given their high price [l-31. As of now, clinically relevant immunosuppression has not been clearly demonstrated for intermediate or high-purity FVIII preparations [l, 4,5]. While this possibility is being further explored there will be a continued need for cost-efficient products of high recovery well into the future. Procedures employing precipitation of impurities by glycine, either solid or in solution [&lo], have been known for some time and these methods have been reported to yield a high recovery of FVIII activity (FVII1:C) and to be of acceptable purity. Recently, a small-scale procedure for the preparation of FVIII was described [ll], based on modifications of a previously reported procedure using glycine and sodium chloride (NaCl) as precipitants [8]. This procedure gave a good recovery of FVII1:C (280 IU/l), at a specif-
ic activity of 10 IU/mg protein and resulted in a stable preparation both in liquid state and during severe dry-heat (8OOC) treatment [ll]. However, the procedure was not scaled up to a clinically useful production size and uncertainty remained in its suitability for use with industrial scale equipment. Additionally, the careful batch-thawing and cryoprecipitate collection described by Brodniewicz-Proba et al. [ll]could not be easily duplicated on a large scale, and it was not known whether cryoprecipitate collected on an industrial scale could be processed to FVIII in a similar manner. Finally, although this preparation was shown to be suitable for the clinically promising [Elsevere dry-heat treatment, its suitability for virus inactivation by more widely accepted methods such as pasteurization [lo] or solventdetergent ( S K I ) treatment [13] was not evaluated. The objectives of the present study were to scale-up the modified glycine precipitation procedure for the preparation of FVIII and to incorporate the S / D virus inactivation technique. The work described here allowed large-scale preparation of an economical, virus-inactivated FVIII concentrate.
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Materials and Methods The starting material for the preparation of FVIII was fresh-frozen plasma from volunteer donors and was obtained from the three Michigan Regions of the American Red Cross. The plasma was separated from whole blood, rapidly frozen within 15 h of collection and stored at or below -30°C until use.
Buffers Tris Buffer. 0.02 M tris-(hydroxymethy1)aminomethaneadjusted to pH 7.0 with HCI. Glycine Buffer. 2.8 M glycine containing 0.3 M NaCl and 0.025 M Tris adjusted to pH 6.8 with HCI. Redissolving Bufer. 0.02 M trisodium citrate containing 0.005 M calcium chloride, 0.04 M NaCl and 1% sucrose, adjusted to pH 7.1 with HCI. Wash Buffer. 1.0 M glycine containing 15% NaCI, 0.02 M trisodium citrate and 0.005 M calcium chloride.
Fig. 1. Isolation of FVIII concentrate.
Final Buffer. 0.02 M sodium citrate containing 0.04 M glycine, 0.06 M NaCI, 0.005 M calcium chloride and 1.5% sucrose, adjusted to pH 7.1 with HCI. Assays FVI1I:Cwas determined by a one-stage automated activated partial thromboplastin time technique on a Lancer Coagulyzer I1 (Sherwood Medical, St. Louis, Mo., USA), using an intermediate-purity FVIII reference standard (Mega-I, 10.2 IU/ml, USFDA) and congenitally FVIII-deficientplasma (George King, Overland Park, Kans., USA) as substrate. FVIII-related antigen (vWf Ag), albumin and fibronectin were measured by rocket immunoelectrophoresisaccording to Laurel1 [14]. Total protein was determind by the biuret method [15], using human albumin as a protein standard. Immunoglobulin gamma (IgG) was measured by radial immunodiffusion. Clottable fibrinogen was determined by a modification of the Clauss method [16]. Tri(n-buty1)phosphate (TNBP) and Ween 80 (T80) were quantitated by gas-chromatographic and spectrophotometric procedures respectively as recommended by Horowitz et al. [13]. Sodium was determined by flame photometry. The absence of any thrombin was ascertained by incubating samples of FVIII concentrate with a 1% solution of fibrinogen in plastic tubes at room temperature for 24 h and observing no sign of clot from intrinsic fibrinogen [17]. Reconstitution time was measured by adding sterile water for injection and gently shaking the vial until no particles were visible. The rabbit pyrogen test was conducted according to prescribed methods [18]. The dose was 1.0 mi (40-50 IU) of reconsituted FVIII concentrate per kilogram of body weight and the results were reported as the sum of the temperature increase of the three rabbits in each test. Preparation Procedure Isolation of FVII1:C. Individual units of plasma sufficient for pools of 620-1,480 liters were removed from -30°C storage and allowed to equilibrate at -10°C overnight. Units of plasma were extruded from satellite bags, crushed, pooled and thawed by the contiuous-thaw method [19]. Cryoprecipitate was harvested in Sharpies AS-16 centrifuges (Pennwalt Corp. Warminster, Pa., USA) in the continuous-
flow mode with supernatant temperature controlled at 0-+2"C. The precipitate was then redissolved in Tris buffer in the proportion of 30 mM of starting plasma, at a temperature of 28-30°C. Glycine buffer, enriched with a 2% aluminum hydroxide suspension (Rehsorptar, Armour), was then added to final concentrations of about 2.0 M glycine and 6.7 ml A1(OH)3per liter of starting plasma. After brief mixing and standing periods, this suspension was centrifuged in a Westfalia centrifuge (Centrico, Inc., Engelwood, N.J., USA) in the continuous-flow mode with supernatant temperature controlled at 28-34°C. Pilot and production scale batches utilized the Westfalia models KO2006 and KDD605 centrifuges, respectively. Granular NaCl was then added to the supernatant to a final concentration of 10% under gentle mixing at a temperature of 28-30°C. After a brief standing period, this suspension was centrifuged in Sharples AS-16 centrifugeswith supernatant temperature controlled at 25-32°C. The NaCl precipitate thus obtained was rapidly frozen and stored at -70°C. The flow diagram for this portion of the process is depicted in figure 1.
viral Inactivation and SID Removal. For viral inactivation, from one to four individual batches of frozen NaCl precipitate were removed from storage and added to redissolving buffer in the proportion of 4.0 ml buffer per liter of starting plasma. The precipitate was dissolved with vibromixing and brought to a temperature of 28-30°C. To remove particulates that may affect the efficacy of viral inactivation, this solution was clarified first by batchwise centrifugation in a Sorvall RC2B centrifuge (DuPont Co., Wilmington, Del., USA) and then by filtration through a 0.45ym filter. After pH adjustement to 6.95-7.0, TNBP and T80 (both from Fisher Scientific) were added to final concentrations of 0.3 and LO%, respectively, followed by incubation for 6 h at 28-30°C under gentle stirring. At the conclusion of incubation, the volume was doubled by the addition of redissolvingbuffer. Precipitation was accomplishedby addition of granular glycine and NaCl under vibromixing to achieve final concentrations of 1.0 M and 15%. respectively. After a brief standing
Preparation of Virus-Inactivated Factor VIII Concentrate
Fig. 2. Flow diagram for the virus inactivation, S / D removal and final FVIII preparation.
period at 28-30°C, the suspension was centrifuged batchwise in a Sorvall RC3B (DuPont Co. Wilmington, Del., USA) for 30 min at 6,000 g. The supernatant was then decanted and the precipitate was thoroughly rinsed with the wash buffer. After dissolving the precipitate in the redissolving buffer at a proportion of 8.0 mM of starting plasma, precipitation was repeated. The second precipitate was redissolved in the final buffer at a proportion of 4.0 mM of plasma. The solution was then diafiltered against lvol of the final buffer in a Benchmark@ rotary ultrafiltration unit (Membrex, Inc., Garfield, N.J.,USA), supplied with a membrane constructed of surface-modified polyacrylonitrile and of 100,OOO molecular weight cut-off. After sterile filtration through a 0.22-pn filter, the final preparation of FVIII was dispensed into 50-ml vials in IO-ml volumes and lyophilized in a Hull Model 24FS40C Lyophilizer (Hull C o p , Hatboro, Pa., USA) at a product temperature below -30°C during primary drying and +25"C during secondary drying. The flow diagram for the virus inactivation, S / D removal and final FVIII preparation is depicted in figure 2.
Results Scale- Up Studies Preliminary pilot-scale experiments were conducted to examine the feasibility of the glycine procedure [ll] with the cryoprecipitate collected by large-scale techniques, in our case by the continuous-thaw method. The results were disappointing, with a mean FVII1:C recovery of 220 IUA plasma and specific activity of 1.9 IU/mg protein (n = 5 ) . The low specific activity prompted an evaluation of the
143
Fig.3. Recovery of FVI1I:C and specific activity in the sodium chloride precipitate at different levels of aluminum hydroxide in the combined aluminum hydroxide/glycine step. Plotted values represent the mean of 3 experiments.
addition of an Al(OH)3 adsorption step. Various levels of Al(OH)3 were evaluated either as a separate step before glycine precipitation or combined into a single step (data not shown). A greater improvement in specific activity was obtained in the combined step, so a series of experiments was undertaken to determine the optimal A1(OH)3 concentration for the combined adsorptiodprecipitation step. The results of this evaluation are illustrated in figure3. The optimal level of Al(OH)3was found to be about 6.7 ml of a 2% suspension per liter of starting plasma. Below this level, specific activity diminished while above this level, FVII1:C recovery rapidly deteriorated with no significant improvement in specific activity. The method for separation of the FVII1:C-rich supernatant from the combined Al(0H)dglycine suspension by filtering through glass beads as described for the small-scale procedure [ll]was not suitable for large-scale application. Based on the amount of glass beads described, the weight of glass beads required for 1,OOO liters starting plasma was estimated to 440 kg. In addition, preliminary experimentation revealed filtration to be slow and that a significant amount of FVIII-rich liquid was lost in the column and in the loosely formed precipitate. As an alternative, two types of continuous-flow centrifuges were evaluated. The Sharples centrifuge created a problem as it caused a 65% loss in the FVII1:C activity presumably due to heavy foaming of the supernatant. On the other hand, continuous-flow centrifugation with the Westfalia centrifuges proved most use-
MyerslWickerhauser/Charamella/Simon/Nummy~rodniewicz-Proba
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Table 1. Pilot (150-200 liters plasma) and production scale (500-620 liters plasma) purification of FVIII
Stage
Cryoprecipitate Glycine/A1(OH)3supernatant NaCl precipitate
FVIII recovery IUA plasma
Total protein recovery mgh plasma
FVIII specific activity IU/mg protein
pilot
production
pilot
production
pilot
production
452k45 307f31 254k39
409 f68 288f32 230f30
1460f181 794k116 67k15
1375f177 808f47 61k11
0.31+0.02 0.37f0.10 4.0fl.l
0.30f0.05 0.38fO.08 3.9f0.7
Results are means f l SD; n = 10 for pilot and n = 10 for production scale.
ful and resulted in minimal loss of VII1:C activity. The supernatant at this step could be separated in production lots from 620 liters starting plasma in 30 min, with a flow rate of about 200 litersh. Mean recovery of FVII1:C activity and specific activity for ten pilot and ten production scale batches are shown in table 1. Overall, FVII1:C recovery was identical (56%) for pilot and production scale batches, based on the starting activity in the cryoprecipitate. Total protein recovery across the two steps was also similar, yielding 4.6 vs. 4.4% for pilot and production scale batches, respectively, based on the total protein in the cryoprecipitate. Activity of FVII1:C in the NaCl precipitate fraction was found to be stable for several months when rapidly frozen and stored at -70°C. The NaCl precipitate averaged 0.70 g/1 of starting plasma. Because of its stability and small mass, this fraction provided a convenient stopping point in the production process and made possible the viral inactivation and final preparation of large batches without the need for increasing the size of the equipment used to obtain the NaCl precipitate from plasma.
SID Reagent Removal Studies Primary focus on these experiments was to develop a reliable method for removal of TNBP and T80. Precipitation was chosen as the method of removal and experiments were designed to determine ways to optimize recovery of FVII1:C while at the same time reducing TNBP and T80 to clinically acceptable levels. In preliminary experiments, it was established that a single precipitation with glycine and NaCl was insufficient for removal of TNBP and T80. Under the conditions of optimal FVII1:C recovery, a single precipitation reduced TNBP and T80 to 350parts per million (ppm) and 1,300ppm, respectively. For further removal of the re-
Fig.4. Effect of glycine and sodium chloride concentrations on recovery of FVI1I:C and residual levels of TNBP and T80 after two successive precipitations. Plotted values represent the mean of 5 experiments.
agents, two successive precipitation steps using various final concentrations of NaCl and glycine were evaluated. The results of these experiments are shown in figure 4. As can be seen, optimal recovery of FVII1:C and maximum reduction of TNBP and T80 were achieved by two successive precipitations at 1.0 M glycine and 15% NaCl. Under these optimized conditions, the levels of TNBP were reduced to 13-22 ppm and those of T80 to 15-35 ppm. Ultrafiltration was then evaluated to further remove TNBP. It was found that TNBP was consistently reduced to a level of 2ppm or less after diafiltration against a single volume of final buffer.
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Preparation of Virus-Inactivated Factor VIII Concentrate
Table 2. Recovery and specific activity of FVIII at various steps of the viral inactivation process Stage
Redissolved, clarified NaCl precipitate Postviral inactivation precipitate 1 Postviral inactivation precipitate 2 Diafiltrated solution Sterile bulk product Final lyophilized product
FV1II:C recovery IUA plasma
Total protein recovery mgll plasma
Specific activity IU/mg protein
239f28
63f12
3.9 f0.8
217f32
57f12
3.9f0.8
218f25 212f20 197f20
52f11 51f11 47fll
4.4f0.9 4.3 f0.9 4.4f0.9
185f17
46f8
4.1f0.6
Results are meanfSD for 9 lots of FVIII.
Several large-scale batches of FVIII were then prepared. Recovery of FVIII:C, total protein and specific activity are summarized in table 2. The relative amounts of FVII1:C activity and protein recovered across each step were similar except for the second precipitation which appeared to result in slight purification. Overall, the FVII1:C recovery for the viral inactivation and S / D reagent removal steps was 89%. Handling losses during sterile filtration were 7% and FVII1:C loss across lyophilization was 6%. Characteristics of the final, virus-inactivated, lyophilized FVIII concentrate for nine large-scale research and clinical trial batches are shown in table 3. The reconstitution time of the product was about 3min with a specific activity of 4 IU/mg of protein and the stability of the reconstituted FVIII (24 h at room temperature) was approximately 90%. Clottable fibrinogen was about 55% of the total protein. Fibronectin, albumin and immunoglobulin gamma were present in much lower concentrations. These individual protein assays accounted for approximately 62% of the total protein present and no attempts were made to identify the remaining proteins. TNBP and T80 were present only in trace amounts of about 1 and 10 ppm, respectively. Processing time at the large-scale required about 4 h to obtain NaCl precipitate from cryoprecipitate, 7 h for redissolving and viral inactivation and 9 additional hours to obtain the sterile filtered final bulk FVIII concentrate. Although steps during most of this 20-hour time period were
Table 3. Characteristics of large-scale produced, S/D-treated FVIII Reconstitution time, s Appearance FVI1I:C IUlml 24-hour reconstituted stability (24 h at 22"C), YO Protein, mg/ml Specific activity, IU/mg protein vWf:Ag, Ulml Clottable fibrinogen, mg/ml Fibronectin, mg/ml Thrombin test (24 h at room temperature) PH Albumin, mg/ml IgG, mg/ml m p PPm n e e n 80, ppm Sodium, mEqA Rabbit pyrogen test, "C
200f63 clear, colorless 44.6f4.4 89f11 11.4f2.4 4.0f0.5 90f29 6.3f2.1 0.69f0.14 negative 6.8fO.09 0.027f0.0068 0.085f0.060 0.97f0.76 11.7f5.8 138f9.3 0.2 f0.2
Results are mean +1SD for 9 lots of FVIII.
conducted at 30°C, rabbit pyrogen test results were consistently acceptable, ranging from no increase to a sum total increase of 0.5 "C in body temperature.
Discussion Much progress in the development of virus-safe, pure FVIII preparations was accomplished in recent years, but it seems that there is still a need for preparation of economical FVIII concentrates [l-31. A recently reported procedure consisting of the preparation of FVIII by modified glycine precipitation, followed by NaCl precipitation resulted in an FVIII preparation of high purity and good recovery [ll]. Although the method was promising, certain aspects of the small-scale production technique were not easily adaptable to large-scale preparation, and a widely accepted virus-inactivation technique had not been incorporated into the process. For these reasons, a study was undertaken to modify the procedure and incorporate a S / D virus inactivation step. Early experiments revealed that the recovery and purity of FVIII obtained by the modified glycine procedure [ll] could not be duplicated using cryoprecipitate collected by large-scale methods. In retrospect, this is not surprising. It is well known that both the recovery of FVII1:C and the level of contaminating proteins in cryoprecipitate may be significantly influenced by the methods for thawing of plasma and collection of cryoprecipitate [20, 211. Subsequent
146
recovery and purification of FVIII may also be affected by the method of thawing and cryoprecipitate collection [19]. The addition of A1(OH)3 to the glycine buffer more than doubled the specific activity of the NaCl precipitate with no additional loss of FVII1:C. The use of an A1(OH)3 adsorption step is not new for conventional techniques for isolation and purification of FVIII [lo, 22-26] but to our knowledge the present study is the first report of combining A1(OH)3 adsorption with the glycine precipitation into a convenient, single step. The diminished FVII1:C recovery at higher concentrations of Al(OHX probably results from a specific adsorption of FVII1:C [25,27]. The successful application of commonly used centrifugation equipment enhances the feasibility of the procedure for large-scale production of FVIII. Its use in the continuous flow mode allows the processing of increased batch sizes without the need to acquire larger equipment and permits this procedure to be easily incorporated into conventional plasma fractionation facilities. The S / D viral inactivation method was chosen because of its demonstrated virucidal capability and its relatively thorough clinical evaluation [2, 28, 291. The nonionic detergent T80 was chosen in lieu of sodium cholate because of the latter’s reported inactivation of FVII1:C during incubation [13]. The redissolved NaCl precipitate fraction was chosen for viral inactivation because of its relatively small volume. This fraction, redissolved as described in the present study, results in a volume of only 4.4 liters for the virus inactivation step from a starting plasma volume of 1,000 liters. This small volume facilitates the performance of virus inactivation and reduces the space and equipment requirements for segregated postviral inactivation-processing steps. The removal of S / D reagents by precipitation was chosen in part for its technical simplicity. Although the development of precipitation techniques required considerable experimentation, they proved to be effective in removing T80 to acceptable levels, overcoming the previously reported failure attributed to micelle formation [13]. Diafiltration was required to further reduce TNBP to levels safely below the acceptable upper limit pf 10ppm (New York Blood Center product specification). Interstingly, there was an over 90% reduction of TNBP with diafiltration against a single volume of the final buffer. This might be due to partial absorption of TNBP to the ultrafilatration membrane. With this S / D reagent removal method, a virus-safe FVIII concentrate is obtained with minimal losses in FVIII:C activity. The final lyophilized product is considered acceptable for clinical use in all respects and is now undergoing clinical evaluation.
MyerslWickerhauser/Charamella/Simon/Nummy~rodniewicz-Proba
References 1 Brettler DB, Levine PH: Factor concentrates for treatment of hemophilia: Which one to choose? Blood 1989;73:2067-2073. 2 Pierce GC, Lusher JM, Brownstein AP, et al: The use of purified clotting factor concentrates in hemophilia. Influence of viral safety, cost and supply on therapy. JAMA 1989;261:3434-3438. 3 Aledort LM, Hilgartner M, Lipton RA, et al: Hemophilia - A treatment in crisis. N Engl J Med 1988;319:1017. 4 Teitel JM, Freedman JJ, Garvey MB, et al: Wo-year evaluation of clinical and laboratory variables of immune function in 117 hemophiliacs seropositive or seronegative for HIV-1.Am J Hematol 1989;32:262-272. 5 Brettler DB: Proposed protocol for the evaluation of the effect of high purity concentrates on the immune system of hemophilia patients. Thromb Haemost 1989;62:811-812. 6 Nilsson IM, Blomback M, Blomback B: Von Willebrand‘s disease in Sweden. Its pathogenesis and treatment. Acta Med Scand 1959;164:263-278, 7 Blomback B, Blomback M: Purification of human and bovine fibrinogen. Arkiv Kemi 1956/57;10:415-443. 8 Thorell L, Blomback B: Purification of the factorVIII complex. Thromb Res 1984;35:431450. 9 Winkelman L, Owen NE, Evans DR, et al: Severely heated therapeutic factor VIII concentrate of high specific activity. Vox Sang 1989$7 :97-103. 10 Heimburger N, Schwinn H, Gratz P, et al: Faktor VIII-Konzentrat, hochgereinigt und in Liisung erhitzt. Arzneimittelforschung 1981;31:619-622. 11 Brodniewicz-Proba T, Beauregard D: Modified glycine precipitation technique for the preparation of factor VIII concentrate of high purity and high stability. Vox Sang 1987;52:10-14. 12 Winkelman L, Feldman PA, Evans DR: Severe heat treatment of lyophilized coagulation factors; in Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Curr Stud Hematol Blood Ttansfus. Basel, Karger, 1989, No 56;pp 55-69. 13 Horowitz B, Wiebe ME, Lippin A, et al: Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-envelopedviruses by tri(n-buty1)phosphate detergent combinations. Transfusion 1985; 25 :5 16-522. 14 Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966;15:45-52. 15 Gornall AG, Bardavill CJ,David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949; 177:751-766. 16 Okuno T, Selenko V Plasma fibrinogen determination by automated thrombin time. Am J Med Tech 1972;38:196-201. 17 Middleton SM, Bennett IH, Smith JK: A therapeutic concentrate of coagulation factors 11, IX and X from citrated, factorVII1-depleted plasma. Vox Sang 1973;24:441456. 18 The United States Pharmacopeia, 21st rev. 1985, pp 1181-1182. 19 Foster PR, Dickson AJ, McQuillan TA, et al: Control of large-scale plasma thawing for recovery of cryoprecipitate factor VIII. Vox Sang 1982;42:180-189. 20 Mason EC, Pepper DS, Griffin B: Production of cryoprecipitate of intermediate purity in a closed thaw-siphon process. Thromb Haemost 1981$6543446. 21 Prowse CV, McGill A: Evaluation of the ‘Mason’ continuous-thaw siphon method for cryoprecipitate production. Vox Sang 1979; 37:235-243.
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22 Wagner RH, Richardson BA, Brinkhous KM: A study of the separation of fibrinogen and antihemophilic factor (AHF) in canine, porcine, and human plasma. Thromb Diath Haemorrh 1957;1:1-8. 23 Hershgold EJ, Pool JG, Pappenhagen AR: The potent antihemophilic globulin concentrate derived from a cold insoluble fraction of human plasma: characterization and further data on preparation and clinical trial. J Lab Clin Med 1966;67:23-32. 24 Newman J, Johnson AJ, Karpatkin MH,et al: Methods for the production of clinically effective intermediate and high-purity factor-VIII concentrates. Br J Haemat 1971;l-20. 25 Wickerhauser M, Mercer JE, Eckenrode Jw: Development of large-scale fractionation methods. VI. An improved method for preparation of antihemophilic factor. Vox Sang 1978;35:18-31. 26 Horowitz B, Lippin A, Chang MY, et al: Preparation of antihemophilic factor and fibronectin from human plasma cryoprecipitate. Transfusion 1984;24:357-362. 27 Seghatchian MJ, Kemball-Cook G, Barrowcliffe TW: Adsorption of factor VIII by aluminum hydroxide. Haemostasis 1979;8:106116.
28 Centers for Disease Control: Safety of therapeutic products used for hemophilia patients. MMWR 1988;37:441450. 29 Horowitz B: Investigations into the application of tri(n-buty1)phosphate/detergent mixtures to blood derivatives; in Morgenthaler JJ (ed): Virus Inactivation in Plasma Products. Curr Stud Hematol Blood Transfus. Basel, Karger, 1989, No 56; pp 83-86.
Received: May 14,1990 Revised manuscript received: September 20,1990 Accepted: October 10,1990 Robert C. Myers, DVM Section of Blood Derivatives Michigan Department of Public Health 3500 N. Logan Street PO Box 30035 Lansing, MI 48909 (USA)
