A small-scale model of factor VIII and factor IX fractionation from plasma

Transfus. Sci. 1989; 10:279-286 Printed in Great Britain

0955-3886189 $3.00+0.00 Pergamon Press plc

A Small-scale Model of Factor VIII and Factor IX Fractionation from Plasma Peter Feldman, BSc, PhD Lowell Winkelman, BA, MSc Helen Evans, BSc Martin Pinnell, HTec Fiona Murdoch, BSc James K. Smith, BSc, PhD

n A small-scale model of factor VIII and factor IX fractionation from human plasma has been developed. Validation experiments demonstrate that it accurately reflects processing at pilot scale. Tests of reproducibility show that there is greater agreement between pools composed of plasma from the same donors than of random donors, but to predict the performance of pilot and manufacturing scale fractionation several pools from different donor panels must be tested. n

tionation would require a considerable speculative investment in terms of both plasma procurement and fractionation. To overcome this problem, we have developed a method for modelling, on a small scale, our factor VIII and factor IX production processes. In this paper we describe the development and validation of this 3-4 kg model (approximately l/1000 of manufacturing scale), the limitations imposed by reduced scale and the controls needed to avoid misinterpretation when comparing data with, and predicting performance at, pilot (300 kg) and manufacturing (3000 kg) scales.

INTRODUCTION

EXPERIMENTAL DESIGN

Plasma procured by Regional Transfusion Centres in England and Wales is fractionated by the Blood Products Laboratory to yield concentrates of factor VIII1 and of factor IX (plus factors II and X),’ followed by ethanol fractionation to produce IgG and albumin. In recent years new systems for collecting plasma by machine apheresis have been introduced and the yield and quality of the blood products derived from such plasmas need to be assessed. At our usual pilot plant scale of 300 kg, evaluation of the suitability of these plasmas and possible variants (handling, anticoagulants etc.) for frac-

The small-scale procedures were designed to model as closely as possible our large-scale fractionation of factor VIII and factor IX concentrates, outlined in Fig. 1. Two main modifications were forced by reduction in scale. Additional insulation of the plasma donations was provided in order to keep the temperature below - 10°C before crushing and thawing and to reduce variation in fibrinogen recovery.3 Also, the size of fractions collected from the factor IX chromatography was modified in order to accommodate the larger bed volumes of ionexchanger-a consequence of the batch centrifugation which replaces the largescale continous-flow centrifuges used at pilot and manufacturing scale; the smallscale fractions, though not identical to

From the Plasma Fractionation Laboratory, Churchill Hos ital, Oxford OX3 7LJ, U.K. (Headquarters: Blood Pro a ucts Laboratory, Elstree, Hem. WD6 3BX, U.K.)

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Thaw at O’C

Extract _ _.

CRY0 SUPERNATANT

II I’

CRY0

EXTRACT

I ]

I]

Adjust pH, temp Add heparin Centrifuge

11 HEPARIN

SUPERNATANT

H,O Adjust pH Add DEAE-cellulose Centrifuge Dtlute +

LOADED GEL

11

I Pack into column Buffer wash Elute with NoCl

Add Glycine/NoCl

EL UATE FRACTIONS Mix fractions by weight

1

I Figure 1.

FACTOR

Vm

]

I

Scheme for the fractionation

those from large-scale batches, related to them in a consistent

FACTOR

IX

I

I

I

I

of factor VIII and factor IX from pooled human

could be manner.

Model Evaluation Initial experiments were performed to determine whether the model could reflect pilot (300 kg) fractionation from the same plasma source and could distinguish between different well-characterized plasma types. Experiments were then performed to establish the sources and range of variation within the small plasma pools. In this paper we describe (i) comparison of factor VIII and factor IX performance in the model and in pilot (300 kg] scale fractionation; (ii) variation between repeated models using plasma pools from the same Region but composed of random donations from different donors; (iii) variation between repeated models using plasma from the same Region in pools made of donations from the same

identified panel”).

donors

(defined

plasma.

as a “donor

Plasma Pools To avoid the introduction of a bias in factor WI recoveries4f5 the 3-4 kg pools were formed whenever possible from approximately equal numbers of blood group 0 and blood group A donations. In this paper all model pools are referred to as “3 kg”. Some plasma pools were fractionated for only the factor VIII or the factor IX processes. The text shows where this occurred by the use of different identification codes for the plasma pools. METHODS Model Fractionation

Method

The factor VIIIprocess. After warming slowly to = - lO”C, frozen plasma donations were stripped, pooled, crushed and thawed manually to 0°C. The cryo-

Small Scale F VIII and FIX

precipitate was recovered (from a cryosuspension in supernatant) by centifugation at 4600 gfor 30 min at 0°C. Cryoprecipitate extraction, heparin precipitation and recovery of the factor VIII by precipitation with glycine and sodium chloride were all carried out as previexcept that where ously described’ continuous centrifugation was used at manufacturing scale, batchwise centrifugation was substituted for the smallscale experiments. The factor fxprocess. After standing at 4°C overnight, 3 kg cryoprecipitate supematant (CPS; diluted l/3 with water and adjusted to pH 6.95) was mixed with DEAE-cellulose (DE 52, Whatman, U.K.; 16.5 g per kg undiluted CPS) for 1 hat 4°C before centrifugation at 4600 g in a Beckman J6 batch centrifuge. The recovered gel was resuspended, packed into a glass chromatography column (i.d. = 22 mm) and washed, at 0.63 mL/min per cm2, with buffer pH 7.0 containing 0.1 M NaCl. The factor IX fractions were eluted with buffer containing 0.25 M NaCl and detected by a rise in eluate absorbance at 280 nm. Fraction sizes were defined as a proportion of the packed gel bed volume. Weighted pools were made from selected fractions. Analytical

Methods

Factor VIII:C was measured by a twostage clotting assay,6 using the 5th and 6th British Working Standards (NIBSC, 85/650 and 87/568) calibrated against the 3rd International Standard for Factor VIII Concentrate 80/556. Samples were assayed on the day of processing without prior freezing and values quoted were a mean of the results obtained by two operators. Fibrinogen was measured as clottable protein by a biuret method.’ Fibronectin was measured by Laurel1 antiimmunoelectrophoresis (Sigma serum), against a standard plasma containing >30 donations. Factor 1X:C activity was measured by a two-stage clotting assay* modified for use with concentrates instead of

281

plasma, using the 1st British Working Standard (NIBSC, 80/560) calibrated against the 1st International Standard for Factor IX Concentrate 72/32. Samples were stored frozen at -40°C before assay. Non-activated partial thromboplastin time (NAPTT) was measured by the method of Kingdon et c.11.~ and was performed on unfrozen samples on the day of processing and subsequently on frozen samples. A clotting time of 150 s was the lower limit required for a 1: 10 dilution of sample to pass the test. Fibrinogen clotting times (FCT) were measured at 37°C after mixing equal volumes of undiluted sample and 0.3% w/v human fibrinogen (Kabi, Grade L). A minimum clotting time of greater than 6 h was required for a sample to pass the test, as specified for clinical concentrates in the 1989 European Pharmacopoeia. Evaluation Criteria for the Model The factor VIII model was evaluated by measurement of the recoveries of FVIII:C, fibrinogen and fibronectin at each of three main processing stages: cryoprecipitate extract, heparin supernatant and redissolved factor VIII precipitate. The factor IX model was evaluated by measurement of FIX:C, NAPTT and FCT for each column eluate fraction and also for a weighted pool of these fractions. The NAPTT and FCT were used as indicators of activation and potential thrombogenicity. Yields of coagulant activity were expressed as a function of plasma weight for factor VIII and CPS weight for factor IX as both these values could be compared directly with data already accumulated from routine pilot-scale fractionation. RESULTS (i) Comparison of Model and Pilot Scale Fractionation Stage 1: model vulidation. Table 1 shows data for a direct comparison of

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Table 1.

Comparison of Results of 3 and 300 kg Factor VIII Fractionation Cryoprecipitate

Poor 1

Pool 2 3 kg 300 kg

Pool 3 3 kg 300 kg

496 437’ 311

404 289 240

382 307 262

450 382 354

470 336 333

667 42

792 44

880 65

736 77

938

943

44

40

54

54

71 55

63 53

306 38

292

258

258

368

367

51 6

37 6

72 10

3 kg

300 kg

FVIII:C (IU/kg plasma) Cryoppt extract Heparin s/n Redissolved ppt

450 351 331

Fibrinogen (mg/kg plasma) Cryoppt extract Heparin s/n

Redissolved ppt

from the Same

Suspension

Fibronectin (mg/kg plasma] Cryoppt extract Heparin s/n

Redissolved ppt l

9

61 13

90 9

This value is unusually high compared with previous pilot fractionation experience.

model and pilot fractionation of factor VIII using the same pool of cryo-suspension as the starting material. Three different cryo-suspension pools (1, 2 and 3) were tested. A similar comparison of model and pilot production was made for factor IX. In this case the common starting material was CPS. Three different pools (4, 5 and 6) were tested (Table 2).

A similar comparison for the factor IX process (Table 4) showed that factor IX yield, NAP’TT and FCT had similar ranges at both scales.

Both factor VIII and factor IX models showed good agreement with the pilot fractionation parameters. Stage 2: model vs batch for pools from different RTCs. Several 3 kg pools (random donors) were fractionated from plasma from each of three Regions for which we already have data at the 300 kg scale. The factor VIII results (Table 3) showed good correlation between model and pilot scales for all three proteins of interest.

Five random lo-donation pools from Region A, matched only for blood groups, were fractionated in the factor VIII model. Variation between pools was observed for all three proteins (Table 5). Four random lo-donation pools from Region B were fractionated in the factor IX model. The results (Table 6) showed some variation in the factor IX yield. The NAPTT showed considerable variation between the pools, from a clear pass (165 s) to a clear fail (132 s).

Table 2.

(ii) Variation Between Random Donor Pools from the Same Region

Comparison of Results of 3 and 300 kg Factor IX Fractionation Cryoprecipitate Supematant

Pool 5 3 kg 300 kg

Pool 6 3 kg 300 kg

497

737

487

530

493

200 >6

116 3

2.25 90

ND 125

146 >6

Pool 4

3 kg

300 kg

FIX:C (W/kg CPS)

568

r;J(;f;r

180 >6

IPooIllsl

ND = not determined.

from the Same

Small Scale FVIII and FIX 283 Table 3.

Comparison 3 Regions

ll=

of 3 with

Region A 3 kg 300 kg 5 13-18.

FVIII:C @U/kg plasma) Cryoppt extract Mean 477 Range 367-520 Heparin

VIII Fractionation

Data

Region B 3 kg 300 kg 7 9

for Plasma

from

Region C 3 kg 300 kg 4 9-11’

469 339-543

461 416-563

463 373-516

362 324-385

410 326-480

s/n

Redissolved

Mean Range

383 298-429

395 328-436

379 328-468

374 313-414

303 268-335

341 326-371

ppt Mean Range

359 268-425

346 29 l-442

369 322-424

326 282-404

252 206-292

295 230-335

801 508-1032

928 635-1215

864 649-1081

615 536-672

729 619-980

55 33-64

86 63-135

68 36-105

22 1731

52 36-81

40 23-61

83 54-117

57 32-85

45 32-53

41 31-49

305 205-477

352 273-378

284 238340

242 199-301

265 222-335

Fibrinogen (mg/kg plasma) cryoppt extract Mean 770 Range 539-933 Heparin s/n Mean 48 Range 31-71 Redissolved ppt Mean 53 Range 40-65 Fibronectin (mg/kg plasma) Cryoppt extract Mean 378 Range 293-442 Heparin

300 kg Factor

s/n

Redissolved

Mean Range

48 27-59

65 38-114

76 36-94

65 49-89

28 20-38

50 41-73

ppt Mean Range

8 3-14

3-816

12 4-15

8 5-12

7 5-10

6 4-12

* 18 batches from Region A and 11 batches from Region C were analysed. A few individual stage yields were not available. The smaller number refers to the minimum analysed on such occasions. Table 4.

Comparison 3 Regions

Il= FIX:C (W/kg CPS) Mean Range

of 3 and 300 kg Factor Region A 3 kg 300 kg 6 17

IX Fractionation

Region C 3 kg 300 kg 6 14

Data

for Plasma

from

Region D 3 kg 300 kg 7 5

523 425-601

499 387-613

452 374-554

465 387-511

594 428-763

481 419-558

NAPTT (main fraction)(s) Mean 189 Range 109-218

185 132-241

192 154-239

198 142-254

154 125-212

158 121-239

3->6

4->6

4->6

4.5->6

2.5->6

FCT (pool)(h) Range

3.75->6

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Table 5. Comparison of Factor VIII Fractionation Data for Five Random lo-donation Pools from Region A 104lonation Pool

1

2

3

4

5

Mean *SD

494 414 411

367 298 268

520 395 319

487 429 371

518 378 425

477 f 63 383 +51 359 265

Fibrinogen (mg/kg plasma) Cryoppt extract 933 Heparin s/n 71 Redissolved ppt 65

539 35 41

909 52 62

732 31 40

738 49 59

Fibronectin (mg/kg plasma) Cryoppt extract 372 Heparin s/n 53 Redissolved ppt 8

293 27 3

442 58 14

397 59 5

385 44 8

FVIII:C (W/kg plasma) Cryoppt extract Heparin s/n Redissolved ppt

Table 6. Comparison of Factor IX Fractionation from Region B lo-donation Pool FIX:C (W/kg CPS) NAPTT (pool)(s) FCT Ihl

1

2

3

4

Mean -tSD

595 139 >6

420 165 >6

554 146 >6

452 132 >6

505 +83 145 +14

Table 7. Comparison of Factor VIII Fractionation Each of Two Donor Panels in Region C D2

Panel D D3

VIII:C @l/kg plasma) Cryoppt extract 400 Heparin s/n 324 Redissolved ppt 326

416 331 333

364 320 301

Fibrinogen (mg/kg plasma) Cryoppt extract 492 26 Heparin s/n Redissolved ppt 29

425 28 39

Fibronectin (mg/kg plasma) Cryoppt extract 270 27 Heparin s/n Redissolved ppt 4

263 26 5

lodonation

Pool

Data for 4 Random IO-donation Pools

Dl

Data for Three lo-donation Panel E E3

Mean* SD

El

E2

393 2 37 325 + 6 320 + 17

327 221 229

303 248 272

518 28 32

419 30 58

388 40 42

478

210 26 4

181 23 4

203 25 4

186 9 5

(iii) Variation in Replicate 4 kg Pools from the Same Donors Three replicate 8-donation pools from each of two different sets of donors from Region C were fractionated for factor VIII. The variation between replicates (Table 7) was less than the difference between the two donor panels.

318 242 242

Pools from

Mean* SD 316 + 12 237214 248 f 22

::

Four replicate CPS pools from the same Region A donor panel were fractionated for factor IX. Table 8 shows that even under these conditions the factor IX yield varied within the 450600 U/kg range. The NAPTT range was 152-l 75 s with a reduced standard deviation compared to the random donor pools.

Small Scale F VIII

and FIX

285

Table 8. Comparison of Factor IX Fractionation Data for Four 3 kg Pools from the Same Donor Panel in Region A Same Donor Pool

1

2

3

4

Mean &SD

FIX:C (IU/kg CPS) NAPTT (pool)(s) FCT IFr31Ihl

450 152 >6

580 162 >6

475 172 >6

491 175 >6

499 * 57 165 f 10

DISCUSSION (i) Comparison of Model and Pilot Scale Fractionation Stage 1: model validation. Table 1 showed the factor VIII model in good agreement with the pilot scale with respect to FVIII:C and fibrinogen recoveries. In particular, FVIII:C was markedly lower for Pool 2 and this was seen at both 3 and 300 kg. The slight differences seen in fibronectin yield between model and pilot scale may reflect the observation that removal of this protein at the two processing stages is particularly sensitive to temperature and pH, parameters which are more difficult to control precisely at 3 than at 300 kg. The factor IX process yields at the two scales [Table 2) showed close agreement for two of the pools (4 and 6). Pool 5 gave a higher yield in the model than in the pilot process and the reason remains unclear. The NAPTT also showed good agreement between model and pilot scale and demonstrate the range of NAPTT that can be obtained (< lo&>200 s). The short NAP’M’ of Pool 5 are characteristic of that plasma when fractionated at 300 kg. Thus the model can differentiate between NAPTT which are long, short or borderline (around the 150 s limit] and between FCT which are long or short, and can identify plasmas which, at the 300 kg scale, produce factor IX with each of these properties. Stage 2: model vs batch for pools from different Regions. Data from several models using plasma from a single Region were then compared with preexisting data from 300 kg fractionation of pools from the same Region. This was repeated for three different Regions.

Table 3 demonstrates good agreement between 3 and 300 kg pools for the three proteins of interest in the factor VIII process. In particular, the lower FVIII:C recovery from 300 kg pools of plasma from Region C is also seen in the 3 kg model pools from this grade of plasma. At both scales the range of recoveries was wide, illustrating the need to take a mean of several results. The factor IX model and pilot process data (Table 4) showed similar ranges.

{ii) Variation Between Random Donor Pools from the Same Regions When pools composed of random donations were fractionated for factor VIII (Table 5) there were considerable differences in yields between the pools. Among factors which might contribute to the observed differences are the small pool size, different plasma storage histories and day-to-day processing and assay differences. When pools of random donations were fractionated for factor IX, considerable yield variation was seen (Table 6). However, at pilot and manufacturing scales, processing yields vary between 400 and 600 U/kg so the model yields are not atypical. The NAPTT showed considerable variation between the pools, from a clear pass (165 s) to a clear fail (132 s). In the four models, the shortest NAPTT for an individual eluate fraction (data not shown) ranged from 125 s (a fail) to 183 s (a clear pass). This suggests that several models from different donor panels may be needed before any plasma collection protocol could be reliably evaluated in terms of the potential thrombogenicity of fractionated factor IX.

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(iii) Variation in Replicate 3 kg Pools from the Same Donors When replicate 3 kg pools from the same donors were fractionated for factor VIII (Table 7), the within-panel variation was less than the between-panel variation. This suggests that the wider differences seen between the random donation pools (ii, above) are introduced by the limitation of donation numbers or by storage differences. In the factor IX model the yields were again in the range 450-600 U/kg. this range is also seen at the pilot scale. However, the NAPTT fell within a closer range ( 152-175 s) than when random donor pools were fractionated. This narrower range is comparable with the dayto-day variation of the NAPTT test (unpublished data). From (ii) and (iii] we infer that the NAPTT of factor IX can vary with the donor composition of the starting pool.

CONCLUSION Large-scale fractionation of plasma to yield factor VIII and factor IX can be modelled on the small scale, requiring only3-4 kgplasma. The data from (ii) and (iii) above suggest that any formal smallscale evaluation of a novel or variant plasma collection procedure should be performed using a pair of plasma pools from the same donor panel, one collected by the “experimental” protocol and one by a well-characterized “control” protocol. Furthermore, as the influence of a single atypical plasma donation in a pool of 10 donations is greater than in a random pool of 600 donations each “variant” and “control” pool should preferably be collected from at least three different donor panels. Well-defined model fractionation will allow economic comparison of novel plasma collection systems with those of known performance. It will also be possible to assess the effects of other factors

(e.g. anticoagulant, freezing rate, plasma storage and handling) on the yield and quality of factor VIII and factor IX after fractionation. Several collaborative studies addressing these questions are in progress and preliminary results are reported in accompanying papers.

Acknowledgements We thank DI David Collms. Dr An elo Robinson ond the staff ot Oxford and Leeds Regon 3 Transfusion Centres for the collection of the matched-donor vools ond also all staff m our prod&tion and assay loborotories whose efforts to maximize samples and assays ot the small stole have resulted in the data we present here.

REFERENCES 1. Winkelman L, Owen NE, Evans DR, Evans H, Haddon ME, Smith JK, Prince PJ, Williams J, Lane RS: Severely heated therapeutic factor VIII concentrate of high specific activity. VOXSang 1989; 57:97-103. 2. Dike GWR, Bidwell E, Rizza CR: The preparation and clinical use of a concentrate containing factor IX, prothrombin and factor X, and a separate concentrate containing factor VII. Br / Haematol 1972; 22:469490. 3. Winkelman L, Pinnell M: Cryoprecipitate composition as a function of plasma softening. Thromb Haemostas 1988; 581338. 4. Preston AE, Barr A: The plasma concentration of factor VIII in the normal population. Br J Haematol 1964; 10238-245. 5. Jeremic M, Weisert 0, Gedde-Dahl TW: Factor VIII (AHG) levels in 1016 regular blood donors. Stand 1 Clin Lab Invest 1976; 36:461~66. 6. Biggs R, Eveling J, Richard G: The assay of antihaemophilic globulin activity. Br \ Haematol 1955; 1:2@34. 7. Coma11 AG, Bardawill CJ, David MM: Determination of serum protein by means of the biuret reaction. I Biol Chem 1949; 177:751-766. 8. Austen DEG, Rhymes IL: A Laboratory Manual of Blood Coagulation. Oxford, Blackwell, 1975, pp. 5658. 9. Kingdon HS, Lundblad RL, Veltkamp JJ, Aronson DL: Potentially thrombogenic materials in factor IX concentrates. Thromb Diath Haemorrh 1975; 33:617631.