Original Papers

0 1991 S. Karger AG, Basel Vox Sang 1991;60:141-147 M)42-9007/9y0603-0141$2.75/0

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 (4 IU/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 immuno- suppression has not been clearly demonstrated for interme- diate or high-purity FVIII preparations [l, 4,5]. While this possibility is being further explored there will be a contin- ued need for cost-efficient products of high recovery well into the future.

Procedures employing precipitation of impurities by gly- cine, 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 ac- ceptable purity. Recently, a small-scale procedure for the preparation of FVIII was described [ll], based on mod- ifications of a previously reported procedure using glycine and sodium chloride (NaCl) as precipitants [8]. This proce- dure 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 prep- aration 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 uncer- tainty 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 [El severe dry-heat treatment, its suitability for virus inactivation by more wide- ly accepted methods such as pasteurization [lo] or solvent- detergent ( 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 prepara- tion 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 con- centrate.

142 Myers/Wickerhauser/Charamell~Simo~ummy~rodniewicz-Proba

Materials and Methods

The starting material for the preparation of FVIII was fresh-frozen plasma from volunteer donors and was obtained from the three Michi- gan 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)aminomethane adjusted 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.

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:C was 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-deficient plasma (George King, Overland Park, Kans., USA) as substrate.

FVIII-related antigen (vWf Ag), albumin and fibronectin were measured by rocket immunoelectrophoresis according 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 deter- mined by a modification of the Clauss method [16]. Tri(n-buty1)phos- phate (TNBP) and Ween 80 (T80) were quantitated by gas-chroma- tographic and spectrophotometric procedures respectively as recom- mended by Horowitz et al. [13]. Sodium was determined by flame photometry. The absence of any thrombin was ascertained by incubat- ing 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 reconsi- tuted 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 cen- trifuges (Pennwalt Corp. Warminster, Pa., USA) in the continuous-

Fig. 1. Isolation of FVIII concentrate.

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, Ar- mour), was then added to final concentrations of about 2.0 M glycine and 6.7 ml A1(OH)3 per liter of starting plasma. After brief mixing and standing periods, this suspension was centrifuged in a Westfalia cen- trifuge (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 centrifuges with supernatant temper- ature 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 solu- tion was clarified first by batchwise centrifugation in a Sorvall RC2B centrifuge (DuPont Co., Wilmington, Del., USA) and then by fil- tration through a 0.45ym filter. After pH adjustement to 6.95-7.0, TNBP and T80 (both from Fisher Scientific) were added to final con- centrations 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 redissolving buffer. Precipitation was accomplished by addi- tion 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 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.

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 Sor- vall 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, precip- itation 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 Lyo- philizer (Hull C o p , Hatboro, Pa., USA) at a product temperature below -30°C during primary drying and +25"C during secondary dry- ing.

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

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 concen- tration for the combined adsorptiodprecipitation step. The results of this evaluation are illustrated in figure3. The optimal level of Al(OH)3 was 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 improve- ment in specific activity.

The method for separation of the FVII1:C-rich super- natant 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 experimenta- tion 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 Sharp- les 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 cen- trifugation with the Westfalia centrifuges proved most use-

144 MyerslWickerhauser/Charamella/Simon/Nummy~rodniewicz-Proba

Table 1. Pilot (150-200 liters plasma) and production scale (500-620 liters plasma) purification of FVIII

Stage FVIII recovery Total protein recovery FVIII specific activity IUA plasma mgh plasma IU/mg protein

pilot production pilot production pilot production

Cryoprecipitate 452k45 409 f 68 1460f181 1375f177 0.31+0.02 0.30 f 0.05 Glycine/A1(OH)3 supernatant 307f31 288f32 794k116 808f47 0.37f0.10 0.38 fO.08 NaCl precipitate 254k39 230f30 67k15 61k11 4 . 0 f l . l 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.

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 exper- iments.

SID Reagent Removal Studies Primary focus on these experiments was to develop a

reliable method for removal of TNBP and T80. Precip- itation was chosen as the method of removal and experi- ments were designed to determine ways to optimize recov- ery 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 1,300 ppm, respectively. For further removal of the re-

agents, two successive precipitation steps using various fi- nal 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 reduc- tion 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.

Preparation of Virus-Inactivated Factor VIII Concentrate 145

Table 2. Recovery and specific activity of FVIII at various steps of the viral inactivation process

Stage FV1II:C Total protein Specific recovery recovery activity IUA plasma mgll plasma IU/mg protein

Redissolved, clarified NaCl precipitate 239f28 63f12 3.9 f 0.8

Postviral inactivation precipitate 1 217f32 57f12 3.9f0.8

Postviral inactivation precipitate 2 218f25 52f11 4.4f0.9

Diafiltrated solution 212f20 51f11 4.3 f0.9 Sterile bulk product 197f20 4 7 f l l 4.4f0.9 Final lyophilized

product 185f17 46f8 4.1f0.6

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 6.8 fO.09 0.027f0.0068 0.085f0.060 0.97f0.76 11.7f5.8 138f9.3 0.2 f0.2

Results are meanfSD for 9 lots of FVIII. Results are mean +1 SD for 9 lots of FVIII.

Several large-scale batches of FVIII were then pre- pared. 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 re- agent removal steps was 89%. Handling losses during ster- ile filtration were 7% and FVII1:C loss across lyophil- ization was 6%.

Characteristics of the final, virus-inactivated, lyophil- ized FVIII concentrate for nine large-scale research and clinical trial batches are shown in table 3. The reconstitu- tion time of the product was about 3min with a specific activity of 4 IU/mg of protein and the stability of the recon- stituted FVIII (24 h at room temperature) was approxi- mately 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 pre- sent only in trace amounts of about 1 and 10 ppm, respec- tively.

Processing time at the large-scale required about 4 h to obtain NaCl precipitate from cryoprecipitate, 7 h for redis- solving and viral inactivation and 9 additional hours to obtain the sterile filtered final bulk FVIII concentrate. Al- though steps during most of this 20-hour time period were

conducted at 30°C, rabbit pyrogen test results were consis- tently 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 econom- ical FVIII concentrates [l-31. A recently reported proce- dure consisting of the preparation of FVIII by modified glycine precipitation, followed by NaCl precipitation re- sulted 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 incor- porated 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 plas- ma and collection of cryoprecipitate [20, 211. Subsequent

146 MyerslWickerhauser/Charamella/Simon/Nummy~rodniewicz-Proba

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 centrifu- gation equipment enhances the feasibility of the procedure for large-scale production of FVIII. Its use in the contin- uous 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 con- ventional 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 de- tergent T80 was chosen in lieu of sodium cholate because of the latter’s reported inactivation of FVII1:C during incuba- tion [13]. The redissolved NaCl precipitate fraction was chosen for viral inactivation because of its relatively small volume. This fraction, redissolved as described in the pre- sent study, results in a volume of only 4.4 liters for the virus inactivation step from a starting plasma volume of 1,000 li- ters. This small volume facilitates the performance of virus inactivation and reduces the space and equipment require- ments for segregated postviral inactivation-processing steps.

The removal of S / D reagents by precipitation was cho- sen in part for its technical simplicity. Although the devel- opment of precipitation techniques required considerable experimentation, they proved to be effective in removing T80 to acceptable levels, overcoming the previously report- ed 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 mem- brane. With this S / D reagent removal method, a virus-safe FVIII concentrate is obtained with minimal losses in FVII- I:C activity. The final lyophilized product is considered acceptable for clinical use in all respects and is now under- going clinical evaluation.

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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)