Pilot scale (300 kg) fractionation of plasma for factor VIII and factor IX

Transfus. Sci. 1989; 10:313-319 Printed in Great Britain

09.553886/89 $3.00+0.00 Pergamon Press plc

Pilot Scale (300 kg) Fractionation of Plasma for Factor VIII and Factor IX David Evans, BSc Tracey Walker, BSc Helen Evans, BSc

n Pilot-scale processing of plasma to factor VIII and factor IX concentrates has been used to provide advance information for manufacturing at >3000 kg scale, and to confirm or extend data obtained by model (3 kg) fractionation experiments. It has been applied especially to the validation of apheresed plasma procured by various semi-automated procedures and in different anticoagulants. There are inter-Regional differences in the quality of plasma as measured by the recovery of factor VIII in the cryoprecipitate, but stage yields on further processing are constant. The low concentration of citrate in ACD-B plasma leads to insecure anticoagulation during the early stages of processing, and such plasma is not acceptable for the routine production of factor IX concentrate. The sequence in which other clinically useful proteins are removed from plasma influences the quality of factor IX concentrate, as measured by in vitro tests for activated factors. n

the minimum investment necessary to provide a clear indication of the way that a particular plasma type is likely to behave during the recovery of our factor VIII concentrate (8Y) and our factor IX concentrate (9A). These techniques have been shown’ to predict performance at the 300 kg pilot scale in operation at the Plasma Fractionation Laboratory (PFL). This offers a well characterised and relatively low risk route for the introduction of new plasma variants to the full (3500 kg] manufacturing scale in operation at the Blood Products Laboratory (BPL). Pilot scale manufacturing has four major advantages: The plasma is processed using conditions which can be designed to approach the manufacturing scale process, particularly in those important aspects which are difficult to scale down to 3 kg, e.g. thawing and sterile finishing operations. Pilot scale fractionation offers a method for the continuous quality assessment of “established” plasma types, which would be quite impractical on the 3500 kg scale. If several hundred donors contribute to a fractionation pool, there is no need for the internal control required by the model systems. The plasma is processed under the disciplines of Good Manufacturing Practice, preserving the possibility of releasing valuable product for informative in vivo use. Pilot scale fractionation has been established at PFL for several years,

INTRODUCTION

The development and application of the “model” (3 kg) fractionation systems have been described in preceding papers.1-5 Work at this scale represents From the Plaama Fractionation Laboratory, Churchill Hospital, Oxford OX3 7LJ (Hea uartera: Blood Products Laboratory, Elstree, Herts. WD6d9 BX), U.K. 313

314 Trunsfus.Sci. Vol. 10. No. 4

providing an increasingly comprehensive library of data. Some of these data were published6 in an earlier evaluation of the Haemonetics plasmapheresis systems, and have been updated in the following discussions.

MATERIALS AND METHODS Source Plasma Fractionated,

19861988

This was collected by the Regional Transfusion Centres (RTCS) in England and Wales, and can be classified into two broad categories: plasma collected by automated plasmapheresis, and plasma recovered from whole blood donations (“recovered plasma”). Within these categories, there was a considerable range of collection procedures. (a) Plasmapheresis plasma. This was mostly collected using the various Haemonetics machines, but the Baxter Autopheresis-C system has recently been introduced at some centres. The results of 3 kg model fractionation using the latter machine have been reported in this series,3 and an evaluation at the pilot scale is currently in progress. The Haemonetics PCS and V50 were the most widely used collection systems, but plasma was also collected using the earlier H50 model and the new Ultralite. Many centres were producing both platelet-poor plasma (PPP) and platelet supernatant plasma (PSP). PSP was usually collected by centrifugation of plateletrich plasma, but the “surge” technique was in occasional use at some centres. The time interval before transferring donations into a freezer ranged from several minutes to several hours, depending on local exigencies. Potentially important differences in the rates of freezing and temperature reduction were introduced as a consequence of the variety of freezing techniques in use. These included: cold rooms at -25”C, blast freezers at -6O”C, dry ice/ethanol, and liquid nitrogen (vapour phase). Storage temperatures ranged from -2.5” to -60°C. The most commonly used anti-

coagulant regimes for Haemonetics ma were:

plas-

Citrate Phosphate Dextrose (CPD50) at 1: 16 (1 vol anticoagulant + 15 vols blood) for PPP. Acid Citrate Dextrose (ACD) at 1: 11; or acid CPD at 1: 11 for PSP. Several other anticoagulants were used, including the low-citrate formulation ACD-B (1: 1 l), which was used for PSP at the Mersey Centre. (b) Recovered plasma. This was mostly PPP separated from the red cells within 18 h of collection. In some cases, up to 28 individual donations were pooled into a 5 L pack prior to freezing. A wide range of freezing techniques was used as described above for apheresed plasma. The most commonly used anticoagulant was CPD adenine (CPD-A) at 1:7. Other anticoagulant formulations were used, but these did not include ACD-B. Factor VIII Fractionation

Process (8Y)

8Y is the high purity, dry-heated concentrate which has been produced at PFL and BPL since 1985. The production process has been described’ and is summarised as follows: Plasma is thawed by a continuous method to a temperature approximately -0.5”C to recover cryoprecipitate. This is extracted into a Tris buffer (the cryoprecipitate extract stage in Table l), and a heparin solution is used to precipitate fibrinogen and fibronectin. Factor VIII is then precipitated using glycine and sodium chloride and redissolved in a small volume of buffer. Residual precipitants are removed by chromatography on Sephadex G-25. The de-salted material (the processed stage in Table 1) is pooled with others for further processing, so this is the final stage at which information is available from an individual pilot pool. The factor VIII recovery at any process stage is defined as the amount of factor VIII remaining at the stage relative to the weight of starting plasma. The

Pilot Scafe Fractionation of FVIII and FIX

process yield is the G-25 recovery expressed as a percentage of the cryoprecipitate extract recovery, providing an indication of how well the factor VIII available in the cryoprecipitate from a particular plasma type survives the 8Y process (excluding finishing operations). The 3 kg model factor VIII process does not include the de-salting step, which is associated with a mean stage yield of 95% factor VIII. Factor IX Fractionation

Process (9A)

9A is a dry-heated factor IX concentrate, which also contains factors II and X. The has been production process summarised’ and involves the adsorption of a plasma supernatant using the anion exchanger DEAE-cellulose. During the course of a comprehensive fractionation programme, three possible sequences of operations on the source plasma may precede the 9A process: Group 1: Removal of cryoprecipitate. of cryoprecipitate, Group 2: Removal followed by a modified Cohn Fraction I (FI-C) precipitation (8% ethanol, -3”C, pH 7.1). of cryoprecipitate, Group 3: Removal followed by adsorption with heparin-Sepharose to adsorb antithrombin III, factor XI and some other proteins, prior to removing FI-C. Two tests are routinely used to indicate potential thrombogenicity of DEAE-cellulose eluate fractions (factor IX eluates): the non-activated partial thromboplastin time (NAPTT) which is believed to detect factor IXa and Xa; and the fibrinogen clotting time (FCT) which measures the concentration of thrombin. An NAPTT of 150 s and an FCT of 3 h are the minimum requirements for an eluate to be considered for further processing. Analytical

Methods

Factor VIII (FVIII:C) was measured by a two-stage clotting methods using the British Working Standard (NIBSC) cali-

315

brated against the contemporary International Standard for factor VIII concentrate. Samples were assayed on the day of processing without freezing, and values quoted were a mean of the results obtained by two experienced operators. Non-activated partial thromboplastin time (NAPTT) was measured by the method of Kingdon et a1.9 Samples were either assayed on the day of processing or after freezing at -40°C. Fibrinogen clotting times (FCTJ were measured at 37°C after mixing equal volumes of undiluted sample and 0.3% w/v human fibrinogen (Kabi Grade L). Samples were stored frozen at -40°C before assay. RESULTS Table 1 summarises our experience of making 8Y from different plasma sources over a 36-month period. In the interest of clarity, recovery data are restricted to two stages: the cryoprecipitate extract recovery, and the processed recovery. The mean factor VIII recovery from all Haemonetics batches was approximately 13% higher than the mean factor VIII recovery from all recovered batches at both process stages. The mean factor VIII recovery from Mersey Haemonetics batches was 1011% higher than the mean factor VIII recovery from all Haemonetics batches at both process stages. There were no significant differences in the percentage yield between cryoprecipitate and processed stages between any of these plasma types. Mersey RTC was the only Centre routinely using ACD-B anticoagulant, and the relevant data are tabulated separately in Table 2. Approximately 70% of the Haemonetics plasma produced by this Centre during this period was PSP in ACD-B. The remainder was mostly PPP in CPD. These grades were not fully segregated for pilot processing, and Table 2 summarises the results of making 8Y from pools in which at least 70% of the starting pool by weight was PPP in CPD, and from pools in which at least 74% of the starting pool by weight was PSP in

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Transfus. Sci. Vol. 10, No. 4

Table 1.

Factor VIII Processing

RTC*

No. of Pools

Typet

Mixed 13 centres

26 138

R H

Yorkshire Sheffield Edgware Lewisham Mersey

42 17 16 11 11

H PP PP PP z

Tooting Manchester Oxford E. Anglia Lancaster Wales S. Western N. Eastern

68 6 5 4 4 3 2

H H PP H H PS H

Yields (8Y) by Region. Pilot scale, PFL, 1986-1988

Anticoagulant* Mainly

CPD-A

CPD 50 CPD 50 CPD 50

CPD 50

ACD-A

Cryoppt Extracts Processed§ Process Yield (FVIII:C Recovery W/kg Plasma)7 (“W 403 + 29 454 + 43

283 f 40 320 f 40

70 70

452 + 44 452 + 39 446 + 25 464+21 500 zb 58

325 325 301 319 355

f 37 zb 28 * 31 * 23 + 48

72 72 67 69 71

428 448 f 30 53 435+39 445 + 49 475 435 440 499

314 306 f+ 39 22 319 + 29 304 + 23 320 293 295 326

:?l 73 68 67 67 67 65

Regional Transfusion Centre. t Plasma type: R = recovered from whole blood, PP = Haemonetics platelet-poor plasma, PS = Haemonetics plasma supematant (not “surge”), H = Haemonetics plasma - PP and PS, including “surge” in some cases. $ Anticoagulant: specified only if this was the sole anticoagulant used during this period. 5 See 8Y process summary for definition. 1 Mean + an-’ (n>4). l

Table 2.

Factor VIII Processing

RTC 12 centress Mersey Mersey

Yield (8Y) for Mersey Plasma. Pilot Scale, PFL, 19861988

No. of Pools

Anticoagulant

128 3 7

No ACD-B >70% CPDT >80% ACD-Bl)

Cryoppt Extract+ Processedt Process Yield (PVIII:C Recovery W/kg Plasma)* (W 450 + 39 461 507 f 60

317 rt 32 318 362 + 44

70 69 71

t See 8Y process summary for definition. $ Mean + o”-i (n>4). 5 All Haemonetics batches, but excluding all Mersey batches. 1 % donations in this anticoagulant in the starting plasma pool. l

ACD-B. In four of these seven batches, ACD-B plasma comprised 100% of the starting pool. The mean factor VIII recoveries at both the process stages from the predominantly ACD-B batches from Mersey were 12-14% higher than the mean factor VIII recoveries from Haemonetics batches from all the other Centres, and lO-14% higher than the factor VIII recoveries from Mersey plasma predominantly in CPD. Table 3 summarises our experience of the NAPTT testing of factor IX eluates from both recovered and Haemonetics plasma over a 48-month period. In this

table, NAPTT results have been grouped into four ranges: <130 s (clear fail), 131149 s (borderline fail], 150-l 70 s (borderline pass), >170 s [clear pass). Mersey batches have been excluded from this analysis. Haemonetics batches were generally associated with shorter NAPTT than recovered plasma batches. The introduction of a Fraction I precipitation prior to the factor IX process increased the failure rate in Haemonetics batches from 25 to 42%, but too few batches were processed in this group to assess the significance of this increase.

Pilot Scale Fractionation of FVIU and FIX Table 3.

NAPTT of Factor IX Intermediate.

Plasma Supernatant Adsorbed

CPS (Group 1)t

Source Plasma 5pe Recovered’

All Haemonetics

AT III depleted FI-C supematant (Group 3)t

All Haemonetics Recovered*

Pilot Scale, PFL, 1985-1988

No. of Batches in NAPTT Ranges (and % Total Batches) 13&149 s >130 s 150-170 s >170 s

All Haemonetics

FI-C supematant (Group 2)t

317

Recovered*

7 (9%) 0

OCl

2 (17%) 0

1; ;;;:I

19 (23%) 0 1 (8%)

42 (52%) 10 (83%)

3 (25%) 0

0

6 (50%) 2 (100%)

6 (19%) 0

5 (16%)

7 (23%)

2 (50%)

1 (25%)

* Excluding Mersey ACD-B batches. t See 9.9 process summary.

Prior adsorption of the plasma with immobilised heparin had a marked effect on the NAPTT of Haemonetics batches, increasing the failure rate to 61%, with most of these in the “clear fail” range, < 130 s. There was a suggestion that this fractionation sequence may also adversely affect batches made from recovered plasma. DISCUSSION Factor VIII

Since the last published review6 which covered the period January-June 1985, the mean factor VIII recovery in cryoprecipitate from Haemonetics plasma has decreased by over 8% (from 496 to 454 IU/kg starting plasma). The corresponding recovery from recovered plasma has decreased by only 2% (from 412 to 403 IU/kg), reducing the advantage originally reported for Haemonetics plasma from 20% to just under 13%. A little over half of the remaining advantage can be accounted for by the reduced volumes of anticoagulant used during Haemonetics procedures. This decrease is not due to a change at a single large producer. Data from eight RTCs were included in the last review, and five of these show a decrease of >5%. Only two Centres show an improvement, and at one of these (Mersey), the increase has an identifiable cause-a change in the anticoagulant formulation which is discussed below. With the exception of Mersey, no

large differences between centres are evident in Table 1, but closer analysis, including data from the other processing stages, suggests there are probably significant differences between some Centres. The improvement in the process yield seen since the last review (from 61 to 70% for Haemonetics batches) was due to a process efficiency improvement at the G-25 de-salting step. The effect of anticoagulant on the factor VIII recovery from Haemonetics plasma has not been rigorously investigated on the pilot scale. In general, the formulation of the anticoagulant has not been shown to be as critical as it is to recovered plasma, where CPD or CPD-A are the anticoagulants of choice.‘&” The exception to this was the ACD-B at 1: 11 introduced at the Mersey Centre. This regime resulted in a plasma citrate concentration less than 60% of that provided by CPD at 1:15 or ACD-A at 1:ll. Observations made during pilot processing of the ACD-B plasma suggest that cryoprecipitate from this source may be dangerously close to coagulating: it exhibits a reduced solubility and samples tend to clot spontaneously. The increased factor VIII recovery in these batches suggests a possible link between low plasma citrate and high factor VIII recovery, but this must be qualified by the results of a carefully controlled model study2 showing that the factor VIII recovery from Mersey Haemonetics plasma in a number of anticoagulants is higher than average.

318 Transjus.Sci. Vol. 10, No. 4

Factor IX The tendency of Haemonetics plasma to produce intermediates with short NAPTT was identified in the previous review.6 Since then the incidence appears to have increased, with one in four batches associated with NAPTT <150 s even when the starting material is diluted CPS (group 1). In this group, intermediates with FCT <3 h (indicating the presence of ~0.01 U/mL thrombin) are uncommon, representing approximately 4% of processed batches. The addition of a heparin-Sepharose adsorption step and a FI-C precipitation to the starting supernatant (Group 3) increased the number of FCT failures to 23% as well as having an adverse effect on NAPTT. Heparin has a relatively low specificity and binds a number of plasma proteins, but it may be significant that AT III is significantly depleted by this step. Only one batch made from recovered plasma failed the FCT test during this period. 9A intermediates made from the ACD-B plasma were invariably associated with exceptionally short NAPTT, and frequently with short FCT. An investigation using the factor IX model2 supports the view that imperfect anticoagulation is responsible for these effects, and this anticoagulant regime is no longer being used to collect plasma for fractionation. There is a suggestion of inter-Regional variation; in particular Haemonetics plasma from Tooting appears less likely to produce a 9A intermediate with short NAPTT. Ten batches from this Centre (all made from diluted CPS) were made during the period; nine of these had NAPTT > 170 s, and only one failed. There is still no clear indication of why 9A from Haemonetics plasma should be more susceptible to failing in vitro tests of potential thrombogenicity. In general, this plasma contains a lower concentration of citrate than recovered plasma, and is also prone to a higher level of platelet contamination. Theoretically,

either or both these factors may contribute to the problem, but except in the case of ACD-B, where the citrate level was exceptionally low, there is insufficient evidence available from the pilot scale data to implicate either. Identifying the causes of the thrombogenicity problems and of the suspected regional variation in both BY and 9A will be complicated by the number of overlapping and possible interactive variables which were described. This challenge will provide considerable scope for model scale investigation in the future. Acknowledgements

tion and analytical sections at PPLfor t and consistent standards of work which haveprovide data for this review.

REFERENCES 1. Feldman P, Winkelman L, Evans H, Pinnell M, Murdoch F, Smith JK: A smallscale model of factor VIII and factor IX fractionation from plasma. Trunsfus Sci 1989; 10:279-286. 2. Duguid J, Winkelman L, Feldman P, Brady A-M: The effect of citrate anticoagulants on apheresed plasma. Transfus Sci 1989; 10:287-293 3. Collins DR, Entwistle CC, Feldman PA, Winkelman L, Evans H, Sims GEC: Fractionation of plasma derived from the Baxter Autopheresis-C and Haemonetics PCS. Trunsfus Sci 1989; 10:295-300. 4. Martlew VJ, Robinson AE, Penny A, Winkelman L, Feldman PA: Fractionation of plasma apheresed using a new toroidal centrifugal device, MSE SPC 600. Transfus Sci 1989; 10:301-304. 5. Penny AF, Townley A, Robinson AE, Pinner M, Winkelman L, Feldman PA: Fractionation of plasma recovered from blood collected by metered anticoagulation. Trunsfus Sci 1989; 10:305-309. 6. Evans DR, Robinson AE, Smith JK: Plasma fractionation and machine plasmapheresis. Plasma Ther Transfus Technol 1986; 7:33-40.

7. 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

Pilot Scafe Fractionation of FVIII and FIX 319 high specific activity. VOX Sang 1989; 57:97-103. 8. Biggs R, Eveling J, Richards G: The assay of antihaemophilic globulin activity. Br I Haematol 1955; 1:20-34. 9. Kingdon HS, Lundblad RL, Veltkamp JJ, Aronson DL: Potentially thrombogenic materials in factor IX concentrates. Thromb Diath Haemorrh 1975; 33:617631.

10. Evans DR, Smith JK, Snape TJ: The influence of plasma age and anticoagulant of the factor VIII content of plasma and the yield of an intermediate purity concentrate. Proc 9th Conf National Blood Trunsfusion Service, 1979. 11. Robinson AE, Penny AP, Smith J, Tovey DL: Pilot study for large-scale plasma procurement using automated plasmapheresis. VOXSang 1983; 44:143-150.