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Virus Validation of PH 4-treated Human Immunoglobulin Produced by the Cohn Fractionation Process
BIOLÐMS 0152 --- 26/4 --- MB 21/5/99 Biologicals (1998) 26, 267±276 Article No. bg980148
Virus Validation of pH 4-treated Human Immunoglobulin Products Produced by the Cohn Fractionation Process Octaaf J. M. Bos1, Dominique G. J. Sunye´1, Carina E. F. Nieuweboer1, Frank A. C. van Engelenburg2, Hanneke Schuitemaker2 and Jan Over1* 1 Department of Product and Process Development; 2CLB Virus Safety Services; CLB, Sanquin Blood Supply Foundation, The Netherlands
Abstract. To assess the virus reducing capacity of Cohn’s cold ethanol fractionation process for the production of intravenous (IVIg) and intramuscular (IMIg) immunoglobulin products, and treatment of these products at pH 4, a validation study of virus removal and/or inactivation was performed using both lipid-enveloped viruses [human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and pseudorabies virus (PSR)], and non-lipid-enveloped viruses [(simian virus 40 (SV40) and encephalomyocarditis virus (EMC)]. For the cold ethanol fractionation process, overall reduction factors of 3·0 logs, r2·6 (Q5·5) logs, 4·6 logs, 5·8 logs and r2·6 (Q6·2) logs were found for HIV, BVDV, PSR, SV40 and EMC, respectively. For all tested viruses the precipitation of fraction III from fraction II + III was the most effective step. From the overall reduction factors it appears that cold ethanol fractionation, although capable of reducing viral infectivity to a significant extent, is not sufficient to meet the requirements of regulatory bodies for viral safety of immunoglobulin products. However, pH 4 treatment contributes effectively to the viral safety of the final products. Treatment at pH 4·05 and 37°C for 16 h, as is applied to IVIg, yields reduction factors of r8·4 logs, r4·0 logs, r7·1 logs, 4·8 logs and 1·4 logs for HIV, BVDV, PSR, SV40 and EMC, respectively. The effectiveness of this process step could be enhanced by extending incubation to 40 h at pH 4·25 compared to 16 h at pH 4·05. The extended incubation, as applied in the production of IMIg, yields a reduction of infectivity of SV40 by r5·5 (Q8·0) logs and of EMC by r4·1 (Q7·1) logs. Storage of IMIg, which is formulated as a solution, at 2–8°C also contributes to virus safety. For storage periods of 8 weeks or longer, reduction factors of 2 to 6 logs were found for all viruses, except for BVDV which remained unaffected. These data indicate that the production processes for IVIg and IMIg as described here have sufficient virus reducing capacity to achieve a high margin of virus safety. = 1998 The International Association of Biological Standardization
Introduction Although immunoglobulin products, derived from human plasma by Cohn's cold ethanol fractionation process,1,2 have been regarded as generally safe,3,4 transmission of hepatitis non-A, non-B (NANBH) and hepatitis C by intravenous immunoglobulin (IVIg) has been reported occasionally.5±12 These transmissions were associated with products manufactured by processes that differed in a number of
*To whom correspondence should be addressed: J. Over, Department of Product and Process Development, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. 1045±1056/98/040267 + 10 $30.00/0
aspects, but included in all cases cold ethanol fractionation. This suggests that the Cohn process has only a limited potential to remove or inactivate viral contaminants, if GMP failures are not included as a possible cause of such transmission. Manufacturers of immunoglobulin products have therefore been urged to both validate their production processes for the capability to clear viruses and implement additional virus inactivation steps when no efficacious virus inactivation step appears to be present.13,14 Treatment of intramuscular immunoglobulin products (IMIg) at pH 4, in the presence of a low concentration of pepsin, was originally developed to 7 1998 The International Association of Biological Standardization
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reduce both anti-complementary activity and contaminating enzymes of the contact activation system, in order to render these products suitable for intravenous administration.15 Subsequently, it was shown that such treatment may also improve viral safety of IVIg.16±18 To improve the safety margin of IMIg, we now have introduced pH 4 treatment as a specific virucidal step in the production process of IMIg. This study describes the assessment of the virus reducing capacity of the Cohn fractionation process, as well as that of the pH 4 step in the production processes of both IVIg and IMIg. With respect to low pH treatment, a comparison is made between the virus reducing capacity of incubation at pH 4⋅05 (for 16 h) and pH 4⋅25 (for 30±40 h).
removal of cryoprecipitate removal of prothrombin complex (optional) removal of C1 –esterase inhibitor (optional) Cryo-depleted plasma (8% EtOH, –3 °C, pH 7.4) Suspension I precipitate I Supernatant I (25% EtOH, –5 °C, pH 6.8) Suspension II + III supernatant II + III Precipitate II + III
Suspended precipitate II + III (12% EtOH, –3 °C, pH 5.1) Suspension III precipitate III Supernatant III (25% EtOH, –5 °C, pH 7.3) Suspension II supernatant II
Materials and methods Plasma and ethanol fractionation process Plasma and in-process fractions thereof were derived from the routine production processes for immunoglobulin products of the Sanquin Blood Supply Foundation, CLB division (CLB; formerly known as Central Laboratory of the Netherlands Red Cross Blood Transfusion Service). The starting plasma pool, collected from healthy, nonremunerated volunteer donors, was regularly made up of 5000 donations, which were all tested and found negative for HBsAg and for antibodies against human immunodeficiency virus types 1 and 2 (HIV-1/2), human T-lymphotropic virus types I and II (HTLV-I/II) and hepatitis C virus (HCV). Both IMIg and IVIg products were manufactured from pooled plasma by the cold ethanol fractionation process according to a combination of Oncley's method2 (first part) and that of Kistler and Nitschmann 19 (second part) (Fig. 1). Temperatures were kept within a range of 20⋅5°C in all steps. All precipitates from the ethanol fractionation process were separated by filtration. With the exception of fraction II, filtration of all fractions was facilitated by adding filter aid (Celite; Celite Corp., Denver, CO).20 Virus reduction data for cryoprecipitation and precipitation of Cohn fraction I were kindly made available by the Finnish Red Cross Blood Transfusion Service (FRC; by courtesy of Prof. G. MyllylaÈ). The process conditions for these steps at the FRC were identical to those of the CLB. Moreover, these virus validation studies of the FRC had also been carried out by CLB Virus Safety Services.
albumin
Precipitate II Suspended precipitate II Lyophilized precipitate II Dissolved powder II pH 4.05, 16 h, 37 °C, pepsin
pH 4.25, 40 h, 37 °C, (no pepsin) dia-/ultrafiltration
i.v. immunoglobin
normal i.m. immunoglobulin, hyperimmune immunoglobulins
Figure 1. Flow chart of the plasma fractionation process for immunoglobulin products as applied by CLB.
pH 4 treatment For the production of the IVIg product, fraction II was dissolved in glucose-containing buffer and lyophilized to remove residual ethanol. Following dissolution of the lyophilized powder II in water for injection to a 7±8% (W/W) protein solution, the pH was carefully lowered to 4⋅05 using 0⋅5 m HCl. Following the addition of pepsin (7 mg/l; Fluka, Buchs, Switzerland) the solution was incubated at 37°C for 16 h. Thereafter, the pH was adjusted to 6⋅25. After sterile filtration and filling, the product was freeze-dried. For the production of IMIg, the procedure was identical to that of IVIg, up to the pH 4 step. To
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enhance the virus-reducing capacity of that step, the time of incubation at pH 4⋅05 was prolonged, but this induced significant amounts of aggregates in the product. At pH 4⋅25 however, the immunoglobulin solution could be incubated for up to 40 h at 37°C without significant polymer generation. Therefore, we carried out pH 4 treatment for IMIg at pH 4⋅25 at 37°C for 40 h, in the absence of pepsin. After incubation at pH 4 a diafiltration step was carried out to replace glucose by glycine and to raise the pH to 6⋅85. Thereafter the solution was concentrated to 16% (W/W) protein concentration, filtered sterile and filled in final containers. The final product was stored as a solution at a temperature of 2±8°C. The pH was measured using a meter (WTW, model 501 or 530; Weilheim, Germany) and a combination pH electrode (Russell pH Ltd., Auchtermuchty, U.K.), calibrated daily using calibration buffers of pH 4⋅00 and 7⋅00 at 20°C. In the course of the incubation the pH rose by about 0⋅1 pH units. Down-scaling of manufacturing steps For measurement of the virus-reducing capacity all tested steps were scaled down from production to laboratory scale. To demonstrate that the downscaling of individual steps was valid, process validation was performed to compare parameters such as pH, protein and IgG contents of the fractions obtained during both routine production and the down-scaled process. However, due to the small volumes used in the down-scaled process, it was difficult to apply the same filtration technique for the removal of precipitates as was used in routine production. Therefore, centrifugation instead of filtration was used to recover the precipitates of the down-scaled process. The virus removal effect of both separation techniques was compared in a separate virus validation study with pseudorabies virus (PSR; also known as suid herpesvirus 1) and simian virus 40 (SV40). Virus culturing and infectivity assays To determine the virus-removing or -inactivating capacity of the various process steps, spiking experiments were performed using the lipidenveloped viruses HIV, bovine viral diarrhoea virus (BVDV) and PSR, and the non-lipid-enveloped viruses SV40 and encephalomyocarditis virus (EMC). HIV was studied being a relevant virus,
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whereas BVDV and EMC were tested as specific model viruses for HCV and hepatitis A virus (HAV), respectively. SV40 and PSR were chosen as general model viruses for non-lipid-enveloped and lipidenveloped DNA viruses, respectively. Virus inocula of HIV (strain HTLV-IIIB), BVDV (strain NADL), PSR (strain Bartha K61), SV40 (strain Pa-57) and EMC (strain EMC) were prepared using H9, EBTr, PD5, CV-1 and Vero cells, respectively. MT2 cells were used for HIV cytotoxicity testing and infectivity assays. For the other viruses the cells used for the infectivity assays were identical to those for preparing virus inocula. The virus inocula for spiking of process steps were derived from virus culture supernatants. However, to obtain a high titre HIV inoculum, the culture supernatant was concentrated 100-fold using ultracentrifugation. All starting materials were spiked with maximally 5% (V/V) of inoculum, which was shown not to affect significantly the properties of the respective starting materials. Starting materials of the process steps were examined for cytotoxic effects. Aliquots of serial dilutions of starting material were added to cell cultures and subsequently inspected microscopically for cytotoxic effects. To ensure the accuracy of the infectivity assays, only non-cytotoxic concentrations of starting materials were used. Infectivity of samples was determined using 50% tissue culture infectious dose (TCID50) assays. Fifty ul aliquots (or 0⋅5 ml for HIV) of 12 (or 8 for HIV) 10-fold serial dilutions were tested in quadruplicate in cell culture. Cell cultures which were incubated for 28, 6, 5, 14 or 6 days (HIV, BVDV, PSR, SV40 and EMC, respectively) were inspected for cytopathic effects. For HIV the read-out was confirmed by reverse transcriptase enzyme activity assay.21 To achieve a lower limit of detection of viral infectivity, larger volumes of samples (20 ml at maximum, depending on the cytotoxicity of the sample) were also tested in bulk cultures in duplicate. The detection limit of the TCID50 and bulk culture assays was calculated with the formula: titre [TCID50/ml] = log (1/V) (V, total volume tested; [V] = ml). As the virus validation studies reported here were carried out before 1994, a few guidelines of the Paul Ehrlich Institute relating to virus infectivity assays22 were not met in this study (i.e. 3-fold serial dilutions to be tested in 8-fold, and TCID50 calculation according to the Spearman-KaÈrber formula). As a result of this, the sensitivity of the infectivity assays in this study was about 0⋅5±1 log TCID50 unit instead of R0⋅5 log.
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Table 1. Protein and IgG recovery of Cohn fractionation steps during down-scaled processing and in routine production Down-scaled processing (n = 3) Plasma fraction
Large-scale production (n = 5)
Protein (%) (mean 2 SD)
IgG (%) (mean 2 SD)
Protein (%) (mean 2 SD)
IgG (%) (mean 2 SD)
100 (55 2 4 g/l) 91 2 1 67 2 3 21 2 7 11 2 1 11 2 1
100 (8⋅2 2 0⋅6 g/l) 101 2 11 7 2 1* 88 2 9 70 2 4 18 2 0
100 (56 2 14 g/l) 89 2 9 71 2 11 23 2 7 11 2 2 11 2 3
100 (6⋅4 2 0⋅2 g/l) 89 2 5 3 2 1* 91 2 7 65 2 9 21 2 3
Cryo-depleted plasma Supernatant I Supernatant II + III Precipitate II + III Supernatant III Precipitate III
*Difference statistically significant, P Q 0·001.
Results Experimental design and down-scaling of the IVIg and IMIg production process In this study, down-scaling experiments were performed to make the scale of the virus validation studies representative for the routine production scale. The scale reduction, expressed as percentage of the production scale, varied between 0⋅001% and 0⋅03% dependent on the step tested. Comparing the recovery of total protein and immunoglobulin G (IgG) in various fractionation steps, we did not detect significant differences between the downscaled processing and large-scale production, except for the residual IgG content in the supernatant of
fraction II + III (Table 1). As both the conditions and the performance of the process at small laboratory scale were similar to those at full production scale, the virus reductions found are most likely to be representative for the virus reduction in routine production. When centrifugation of precipitates of the cold ethanol fractionation was compared to filtration with respect to removal of PSR and SV40, no significant differences were found between the two separation techniques for both fraction II + III and fraction III (Table 2). Therefore, the centrifugation in the virus validation experiments is considered to be a representative alternative to the filtration technique used in the large-scale production setting.
Table 2. Viral reduction factors (in log TCID50 units) for PSR and SV40 for the precipitation of fractions II + III and III: comparison between centrifugation and filtration Centrifugation
Fraction II + III: Suspension II + III² Supernatant II + III Precipitate II + III Fraction III: Suspension III² Supernatant III Precipitate III
Filtration
PSR*
SV40*
PSR
SV40
−0⋅6/−0⋅1 2⋅6/r3⋅4 −0⋅3/0⋅2
−0⋅4/−0⋅8 r3⋅4/r2⋅9 0⋅5/−0⋅2
−0⋅1 r3⋅4 −0⋅1
−0⋅8 r3⋅2 −0⋅1
0⋅2/0⋅5 3⋅3/r3⋅9 0⋅8/1⋅0
−0⋅4/0⋅3 2⋅4/2⋅9 −0⋅2/0⋅2
0⋅5 3⋅2 0⋅5
0⋅3 2⋅6 −0⋅1
*The results of two experiments are shown. †Suspensions II + III and III were incubated for 1 h and 2 h respectively, before separation of the precipitates.
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Table 3. Viral reduction factors (in log TCID50 units) of the Cohn fractionation process for fraction II Process step Removal of cryoprecipitate
HIV
BVDV
PSR
SV40
EMC
0⋅2
−0⋅4*
0⋅4
1⋅4
0⋅3*
Fraction I: Suspension I* (1 h/10 h)² Supernatant I* (1 h/10 h) Precipitate I* (1 h/10 h)
1⋅0/ND 0⋅8/0⋅8 1⋅4/1⋅4
0⋅2/ND 0⋅4/0⋅7 0⋅5/0⋅8
0⋅5/ND r2⋅4/1⋅3 −0⋅7/−0⋅1
0⋅8/ND 2⋅0/2⋅6 0⋅9/0⋅4
0⋅3/ND 0⋅0/−0⋅2 0⋅9/1⋅2
Fraction II + III: Suspension II + III (1 h/15 h)² Supernatant II + III (1 h/15 h) Precipitate II + III (1 h/15 h)
0⋅9/ND 1⋅1/2⋅9 R0/R0
0⋅2*/ND 0⋅9*/ND −0⋅2*/ND
−0⋅6/ND 2⋅6/2⋅6 −0⋅3/−0⋅5
−0⋅4/ND r3⋅4/r3⋅4 0⋅5/0⋅3
ND/ND ND/ND ND/ND
Fraction III: Suspension III (2 h/15 h)² Supernatant III (2 h/15 h) Precipitate III (2 h/15 h)
0⋅2/−0⋅1 3⋅5/3⋅0 −1⋅0/ND
0⋅2/0⋅5 r2⋅6/r2⋅6³ 0⋅5/ND
0⋅2/−0⋅1 3⋅3/3⋅5 0⋅8/ND
−0⋅4/−0⋅7 2⋅4/3⋅0 −0⋅2/ND
0⋅2/0⋅2 r2⋅6§/r2⋅8§ −0⋅1/0⋅2
3⋅0
r2⋅6³
4⋅6
5⋅8
r2⋅6§
Overall reduction factor¶
ND: not determined. *Results of virus validation studies carried out by CLB Virus Safety Services on behalf of the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland); process conditions were identical to those of CLB. ²Duration of incubation after reaching the final process conditions for that step and before separation of the precipitate was carried out. One hour and 2 h incubation time, respectively, are the routine conditions. ³ Q 5⋅5; § Q 6⋅2. ¶In accordance with EC guideline EC/8115/89-EN,13 only reduction factors larger than 1⋅0 log (printed in bold) were used to calculate the overall reduction factors.
Virus reduction in the cold ethanol fractionation process The virus-spiking studies of the cold ethanol fractionation process steps leading to fraction II show that for suspensions I, II + III and III no significant reduction of viral infectivity was found in spite of the presence of ethanol concentrations up to 25%, regardless of whether a short (1±2 h) or long (10±15 h) incubation time was applied after achieving the process conditions for inducing the precipitate (Table 3). After separation of the precipitates essentially all virus was recovered in the precipitate. This resulted in some reduction of infectious virus in the corresponding supernatant, supernatant I showing the smallest reduction and supernatant III the largest (Table 3). Therefore, the mechanism of viral reduction in supernatant III is removal rather than inactivation. Virus inactivation by low pH treatment The reduction of virus infectivity in the IVIg preparations treated at pH 4⋅05 or pH 4⋅25 in the presence of pepsin demonstrates that low pH treatment has a significant virus inactivating
capacity (Table 4). Both at pH 4⋅05 and at pH 4⋅25 HIV and PSR were reduced within 16 h to titres below the detection limit. However, BVDV was not reduced below the detectable titer until treated for 30 h at pH 4⋅25 (infectivity still detectable after 16 h when assayed in a bulk culture). The kinetics of virus inactivation at pH 4⋅05 (Fig. 2) show that lipid-enveloped viruses were inactivated more efficiently than non-lipid-enveloped viruses. None of the tested non-lipid-enveloped viruses were inactivated to an undetectable titre within 40 h of treatment at pH 4⋅25 (Table 4). However, the kinetics of inactivation of EMC show that a long incubation period at pH 4⋅25 is more effective than a relatively short incubation at pH 4⋅05 (Fig. 3). This is a result of the duration of incubation rather than to the small difference in pH, as the kinetics of inactivation at both pH values are similar. Reduction of viral infectivity by pH 4⋅25 treatment of IMIg preparations in the absence of pepsin was equivalent to that of IVIg in the presence of pepsin (Table 4). This is also clearly shown by the kinetics of inactivation of EMC (Fig. 3). Thus it seems that the presence of trace amounts of pepsin
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Table 4. Viral reduction factors (in log TCID50 units) of incubation at pH 4⋅05 and pH 4⋅25 for intravenous immunoglobulin preparations (in the presence of pepsin) and intramuscular preparations (in the absence of pepsin) HIV
BVDV
PSR
SV40
EMC
Intravenous immunoglobulin Incubation for 16 h at pH 4⋅05 Incubation for 16 h at pH 4⋅25 Incubation for 40 h at pH 4⋅25
r8⋅4 r7⋅3 r7⋅3
r4⋅0* r3⋅5² r6⋅0
r7⋅1 r8⋅0 r8⋅0
4⋅8 r5.5³ r5⋅5³
1⋅4 2 0⋅3 2⋅6 r4⋅1§
Intramuscular immunoglobulin Incubation for 16 h at pH 4⋅25 Incubation for 40 h at pH 4⋅25
r6⋅8 r6⋅4
r4⋅0* r6⋅5
r6⋅0 q8⋅5
r5⋅4¶ r5⋅4¶
2⋅6 r3⋅6>
* Q 6·5; †Q6·0; ‡Q8·0; §Q7·1; ¶Q7·9; >Q7·2.
(in the case of IVIg) did not contribute significantly to virus reduction during incubation at pH 4⋅25. Virus inactivation during storage Storage of IMIg (which is formulated as a solution) at 2±8°C revealed some virus inactivating potential (Table 5). The extent of inactivation varied with the virus tested, HIV being affected the most, while BVDV showed little or no inactivation at all. It should be emphasized that the presence of thiomersal as an antimicrobial additive to the 15-ml filling of IMIg resulted in the demonstration of somewhat lower reduction factors due to its cytotoxicity in the titration assay: viral infectivity could be assayed only at relatively high test sample dilutions. Therefore, the real reduction by storage in the presence of thiomersal was likely to be similar to that in the absence of thiomersal.
The total reduction factors for the entire production processes for both types of immunoglobulin products are summarized in Table 6. Discussion When an overall capacity of at least 6 logs reduction of model viruses is applied as criterion for processes yielding virus safe products, the data show that the version of Cohn's cold ethanol fractionation process, as is in use at CLB, is unable to ensure virus safety of the immunoglobulin products on its own (Table 3). Precipitation of fraction III is the only process step in which viruses were consistently removed to a significant extent. As a reduction of only about 3 logs was achieved for all tested viruses, this process step cannot be regarded as an effective virus reducing step, for which a reduction of at least 4 logs holds.23 For suspensions I, II + III and III no
9 8
8 EMC
7
6 SV40
5 4
log TCID50
log TCID50
7
BVDV
3
PSR
2
5
pH 4.25 – pepsin
4 3
pH 4.25 + pepsin
2
HIV
1
pH 4.05 + pepsin
6
1 0
5
10 Time (h)
15
20
Figure 2. Kinetics of virus inactivation in IVIg solutions at pH 4⋅05 (pepsin present). Open symbols indicate infectivity titers below the detection limit.
0
5
10
15
20 25 Time (h)
30
35
40
45
Figure 3. Kinetics of inactivation of EMC at three regimes of low pH treatment.
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Table 5. Viral reduction factors (in log TCID50 units) of storage conditions for intramuscular immunoglobulin preparations Storage time
HIV
BVDV
PSR
SV40
EMC
With thiomersal:* 8 weeks 12 weeks 16 weeks
r3⋅7 r3⋅7 r3⋅7
1⋅0/0⋅4 1⋅5/0⋅2 ND/0⋅9
r2⋅3 r2⋅5 r6⋅2
r1⋅6 r1⋅6 r1⋅6²
r2⋅3 r2⋅3 r2⋅3³
Without thiomersal:* 8 weeks 12 weeks 16 weeks
4⋅7 r5⋅3 r6⋅0§
r2⋅7 r3⋅2 r5⋅0¶
r4⋅3 r4⋅3 r4⋅3>
2⋅9 2⋅9 r3⋅4**
1⋅2 0⋅8 0⋅5
ND: not determined. *Thiomersal is added as an anti-microbial agent to the 15 ml multi-dose filling, but not to the 1, 2 and 5 ml fillings of the IMIg products of CLB. ² Q 5⋅1; ³ Q 5⋅8; § Q 6⋅6; ¶ Q 7⋅4; > Q 6⋅7; ** Q 7⋅1.
virus reduction at all (i.e. less than 1 log TCID50) was found. This means that none of the tested viruses were inactivated by ethanol concentrations up to 25% at a temperature of −3°C or −5°C. The results further show that the viruses preferentially go with the precipitates, as has also been found earlier.24±29 The limited virus reducing capacity of the cold ethanol fractionation found in this study is consistent with other reports,26±32 although in more early work a higher virus reducing capacity has been reported, including inactivation by ethanol, e.g. for HIV.24,25,33,34 Part of this discrepancy may be explained by not strictly applying the low temperatures of the cold ethanol fractionation process in the laboratory experiments, as HIV is sensitive to ethanol at temperatures above zero.31 Not all cold fractionation process steps have been validated in
this study, so the actual virus reduction may be larger than that demonstrated in this study. Still, a specific virucidal step seems necessary to ensure viral safety of the immunoglobulin products. A similar conclusion has been drawn by others,7,9,26,28,29,35 some of them referring to the NANBH transmissions reported in the literature. Treatment at pH 4 appears to be an effective virucidal step, as reduction factors for the tested viruses are about 4 logs or higher (Table 4). This finding is in accordance with results published previously.3,16±18 The lack of a synergistic effect exerted by the presence of trace amounts of pepsin is in contrast to previous studies,17,18 where it was shown that pepsin enhances the overall effectiveness of the pH 4 treatment. Part of the discrepancy may be due to the fact that both reported studies
Table 6. Overall viral reduction factors (in log TCID50 units) for intravenous and intramuscular immunoglobulins Process step
HIV
BVDV
PSR
I.v. immunoglobulin Cohn fractionation (Table 3) Incubation for 16 h at pH 4⋅05 (Table 4) Overall reduction factor:
3⋅0 r8⋅4 r11⋅4
r2⋅6* r4⋅0³ r6⋅6
I.m. immunoglobulin Cohn fractionation (Table 3) Incubation for 40 h at pH 4⋅25 (Table 4) Storage for q8 weeks (Table 5) Overall reduction factor:
3⋅0 r6⋅4 4⋅7 r14⋅1
r2⋅6* r6⋅5 Q1⋅0 r9⋅1
* Q 5⋅5; ² Q 6⋅2; ³ Q 6⋅5; § Q 7⋅9; ¶ Q 7⋅2.
SV40
EMC
4⋅6 r7⋅1 r11⋅7
5⋅8 4⋅8 10⋅6
r2⋅6² 1⋅4 r4⋅0
4⋅6 q8⋅5 r2⋅7 q15⋅8
5⋅8 r5⋅4§ r4⋅3 r15⋅5
r2⋅6² r3⋅6¶ 2⋅9 r9⋅1
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were performed at pH 4⋅0 and pH 4⋅05, respectively, while we applied pH 4⋅25. Kempf et al. 18 showed that the synergistic effect of pepsin increases with decreasing pH. Another explanation may be that the model viruses used in the reported studies were not the same as used here. Kempf et al. demonstrated this effect only for the relatively resistant vesicular stomatitis virus (VSV), but not for four other lipid-enveloped viruses tested. In the study of HaÈmaÈlaÈinen et al. 17 Semliki forest virus proved to be sensitive to pepsin, while VSV was rather insensitive. Immunoglobulin for intramuscular administration which has not been treated at pH 4 has never been implicated in transmission of HIV, NANBV, HCV or hepatitis B virus, despite decades of routine clinical use. Although not all production steps possibly contributing to virus inactivation or removal (e.g. freeze-thawing of the precipitates or freeze-drying3,25,29) have been validated in this study, the good clinical record of viral safety of IMIg preparations especially indicates that also factors other than the Cohn fractionation steps must play a role in achieving viral safety. It should be taken into account that virus safety of immunoglobulin products with respect to some relevant viruses, like HAV and parvovirus B19, is most probably increased by immune neutralization due to the presence of significant amounts of antibodies neutralizing HAV and parvovirus B19. Furthermore, the viral safety of intramuscular preparations may be positively influenced by storage of the final product in its liquid form. This study shows that the storage conditions of IMIg preparations at (near-)neutral pH has a virus inactivating effect, although the extent of inactivation depended on the virus tested. This effect of storage has also been described by other groups.3,17,32 Also, other factors contribute to reducing the viral transmission risk, such as the use of plasma from non-remunerated donors, the policy to discourage persons who carry an increased risk for blood-borne viruses (like intravenous drug abusers) to donate, and the screening of all donations for viral markers like anti-HIV-1/2, anti-HCV and HBsAg. Over the years, viral transmission caused by the use of CLB's IVIg and IMIg products has never been reported to us, whereas transmission of NANBH through IVIg products has occurred incidentally elsewhere.5±12 Several possible reasons have been suggested to explain these transmissions. Among these are production in a pilot plant, an excessive viral load in the starting plasma, or GMP failure.7,10
It now turns out that the Cohn fractionation process on its own is insufficient to guarantee virus safety. This is also demonstrated by more recent transmissions of HCV by an intravenous immunoglobulin product which was prepared by Cohn fractionation, but without additional viral reduction methods like organic solvent/detergent treatment or low pH.11 However, transmission of HCV by this product only occurred after the introduction of anti-HCV screening of the plasma donations by second-generation assays,4,12 which are more efficient in detecting antibodies to HCV than the first-generation assays (anti-C100, anti-C33c and anti-core instead of anti-C100 only in the case of the latter36). Most likely, antibodies to HCV originally present in the plasma pool contributed to the viral safety of the product, possibly by formation of complexes with the virus which were diverted away from the final immunoglobulin product in the purification process, while uncomplexed virus is not. This hypothesis was already postulated in 199037 and has led to a study in chimpanzees which showed no risk of transmitting HCV by immunoglobulin products derived from plasma screened for anti-HCV by first-generation assays.38 The transmission of NANBV by a product, prepared by the Cohn fractionation process and treated at pH 4⋅05 for 16 h10 cannot be explained in this way. An exceptionally high viral load of the starting plasma pool or a GMP failure have been postulated as possible causes.10 It should be noted that in this case only one product batch was involved, which later turned out to be negative for HCV-RNA by PCR testing.39 Reconsidering all data available, it may be concluded that extended incubation at pH 4 is capable of effectively inactivating lipid-enveloped viruses. In combination with the limited virus reducing capacity of the Cohn fractionation, pH 4 treatment reduces the risk of transmission of lipid-enveloped viruses by the IVIg product to a minimum. The infectivity of non-lipid-enveloped model viruses is less readily reduced below the detection limit by low pH treatment, which urges prolonged treatment (up to 40 h) at low pH. With respect to relevant non-enveloped viruses, immune neutralization also contributes to viral safety, while for IMIg some additional virus reduction is obtained by the storage condition of the liquid final product. The latest safety measure that has now become possible is screening of the starting plasma for the presence of viral DNA or RNA of relevant viruses by the PCR technique,40,41 as has been in use in our
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centre for HCV since late 1995. As a result of all these measures IVIg and IMIg products are obtained which can be considered to carry a minimal risk of virus transmission. Acknowledgements We thank Prof. G. MyllylaÈ of the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland, for making available part of the virus validation data of the cryoprecipitation and Cohn fractionation processes. Prof. W. G. van Aken (CLB) is gratefully acknowledged for his constructive criticism on this paper. References 1. Cohn EJ, Strong LE, Hughes WL Jr et al. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946; 68: 459±475. 2. Oncley JL, Melin M, Richert DA et al. The separation of the antibodies, isoagglutinins, prothrombin, plasminogen and b1-lipoprotein into subfractions of human plasma. J Am Chem Soc 1949; 71: 541±550. 3. Cuthbertson B, Perry RJ, Foster PR et al. The viral safety of intravenous immunoglobulin. J Infection 1987; 15: 125±133. 4. Yap PL. The viral safety of intravenous immune globulin. Clin Exp Immunol 1996; 104 (Suppl 1): 35±42. 5. Lane RS. Non-A, non-B hepatitis from intravenous immunoglobulin. Lancet 1983; ii: 974±975. 6. Lever AML, Webster ADB, Brown D et al. Non-A, non-B hepatitis occurring in agammaglobulinaemic patients after intravenous immunoglobulin. Lancet 1984; ii: 1062±1064. 7. Ochs HD, Fischer SH, Virant FS et al. Non-A, non-B hepatitis and intravenous immunoglobulin. Lancet 1985; i: 404±405. 8. Weiland O, Mattson L, Glaumann H. Non-A, non-B hepatitis after intravenous gammaglobulin. Lancet 1986; i: 976±977. 9. BjoÈrkander J, Cunningham-Rundles C, Lundin P et al. Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinemia or IgG subclass deficiency. Am J Med 1988; 84: 107±111. 10. Williams PE, Yap PL, Gillon J et al. Transmission of non-A, non-B hepatitis by pH 4-treated intravenous immunoglobulin. Vox Sang 1989; 57: 15±18. 11. Anonymous. Outbreak of hepatitis C associated with intravenous immunoglobulin administrationÐUnited States, October 1993±June 1994. Morb Mort Wkly Report 1994; 43: 505±509. 12. Yu MW, Mason BL, Guo ZP et al. Hepatitis C transmission associated with intravenous immunoglobulins. Lancet 1995; i: 1173±1174. 13. EEC Regulatory Document. Note for Guidance: Validation of virus removal and inactivation procedures. Biologicals 1991; 19: 247±251.
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14. Ad hoc Working Party on Biotechnology/Pharmacy of the CPMP. Blood products and non-enveloped viruses. III/5830/93 Final. 15. Friedli HR. Methodology and safety considerations in the production of an intravenous immunoglobulin preparation. Pharmacotherapy 1987; 7 (Suppl.): 36S±40S. 16. Reid KG, Cuthbertson B, Jones ADL et al. Potential contribution of mild pepsin treatment at pH 4 to the viral safety of human immunoglobulin products. Vox Sang 1988; 55: 75±80. 17. HaÈmaÈlaÈinen E, Suomela H, Ukkonen P. Virus inactivation during intravenous immunoglobulin production. Vox Sang 1992; 63: 6±11. 18. Kempf C, Jentsch P, Poirier B et al. Virus inactivation during production of intravenous immunoglobulin. Transfusion 1991; 31: 423±427. 19. Kistler P, Nitschmann H. Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier. Vox Sang 1962; 7: 414±424. 20. De Jonge E, Van Leeuwen MAW, Radema H et al. Filtration processes in the Cohn fractionation. In: Rivat C, Stoltz JF, (eds). Biotechnology of Blood Proteins, London: Colloque INSERM/John Libbey Eurotext Ltd., 1993; Vol. 227: 49±54. 21. Tersmette M, Van Dongen JJM, Clapham PR et al. Human immunodeficiency virus infection studies in CD-4 expressing human-murine T-cell hybrids. Virology 1989; 168: 267±273. 22. Bundesgesundheitsamt und Paul-Ehrlich-Institut. Bekanntmachung uÈber die Zulassung von Arzneimitteln; Anforderungen an Validierungsstudien zum Nachweis der Virussicherheit von Arzneimitteln aus menschlichen Blut oder Plasma vom 20. Dezember 1993/21. Januar 1994. Bundesanzeiger 1994; 4742±4744. 23. Note for Guidance on Virus Validation Studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses. (CPMP/BWP/268/95). 24. Wells M, Wittek AE, Epstein JS et al. Inactivation and partition of human T-cell lymphotropic virus, type III, during ethanol fractionation of plasma. Transfusion 1986; 26: 210±213. 25. HeÂnin Y, MareÂchal V, BarreÂ-Sinoussi F et al. Inactivation and partition of human immunodeficiency virus during Kistler and Nitschmann fractionation of human blood plasma. Vox Sang 1988; 54: 78±83. 26. Yei S, Yu MW, Tankersley DL. Partitioning of hepatitis C virus during Cohn-Oncley fractionation of plasma. Transfusion 1992; 32: 824±828. 27. Louie RE, Galloway CJ, Dumas ML et al. Inactivation of hepatitis C virus in low pH intravenous immunoglobulin. Biologicals 1994; 22: 13±19. 28. Willkommen H, Scheiblauer H, LoÈwer J. Partitioning and inactivation of viruses during the ethanol fractionation of plasma. Abstract Symposium on Viral Blood Safety and Screening, Washington D.C., October 1994. 29. DichtelmuÈller H, Rudnick D, Breuer B et al. Validation of virus inactivation and removal for the manufacturing procedure of two immunoglobulins
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30. 31. 32. 33.
34.
35. 36.
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and a 5% serum protein solution treated with b-propiolactone. Biologicals 1993; 21: 259±268. Prince AM, PieÈt MPJ, Horowitz B. Effect of Cohn fractionation conditions on infectivity of the AIDS virus. N Engl J Med 1986; 314: 386±387. Piszkiewicz D, Kingdon H, Apfelzweig R et al. Inactivation of HTLV-III/LAV during plasma fractionation. Lancet 1985; ii: 1188±1189. Mitra G, Wong MF, Mozen MM et al. Elimination of infectious retrovirus during preparation of immunoglobulins. Transfusion 1986; 26: 394±397. Hamamoto Y, Harada S, Yamamoto N et al. Elimination of viruses (Human Immunodeficiency, Hepatitis B, Vesicular Stomatitis and Sindbis viruses) from an intravenous immunoglobulin preparation. Vox Sang 1987; 53: 65±69. Grandgeorge M, Pelloquin F. Inactivation of the human immunodeficiency viruses (HIV-1 and HIV-2) during the manufacturing of placental albumin and gammaglobulins. Transfusion 1989; 29: 629±634. Suomela H. Inactivation of viruses in blood and plasma products. Transf Med Rev 1993; II: 42±57. Bresters D, Cuypers HTM, Reesink HW et al. Enhanced sensitivity of a second generation ELISA
37. 38.
39. 40. 41.
for antibody to hepatitis C virus. Vox Sang 1992; 62: 213±217. Finlayson JS, Tankersley DL. Anti-HCV screening and plasma fractionation: the case against. Lancet 1990; i: 1274±1275. Biswas RM, Nediar S, Wilson LT et al. The effect on the safety of intravenous immunoglobulin of testing plasma for antibody to hepatitis C. Transfusion 1994; 34: 100±104. Yap PL, McOmish F, Webster ADB et al. Hepatitis C virus transmission by intravenous immunoglobulin. J Hepatol 1994; 21: 455±460. Saldanha J, Minor P. A sensitive PCR method for detecting HCV RNA in plasma pools, blood products, and single donations. J Med Virol 1994; 43: 72±76. Saldanha J, Minor P. Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture. Br J Haematol 1996; 93: 714±719.
Received for publication 8 April 1998; accepted 20 July 1998
Virus Validation of pH 4-treated Human Immunoglobulin Products Produced by the Cohn Fractionation Process Octaaf J. M. Bos1, Dominique G. J. Sunye´1, Carina E. F. Nieuweboer1, Frank A. C. van Engelenburg2, Hanneke Schuitemaker2 and Jan Over1* 1 Department of Product and Process Development; 2CLB Virus Safety Services; CLB, Sanquin Blood Supply Foundation, The Netherlands
Abstract. To assess the virus reducing capacity of Cohn’s cold ethanol fractionation process for the production of intravenous (IVIg) and intramuscular (IMIg) immunoglobulin products, and treatment of these products at pH 4, a validation study of virus removal and/or inactivation was performed using both lipid-enveloped viruses [human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and pseudorabies virus (PSR)], and non-lipid-enveloped viruses [(simian virus 40 (SV40) and encephalomyocarditis virus (EMC)]. For the cold ethanol fractionation process, overall reduction factors of 3·0 logs, r2·6 (Q5·5) logs, 4·6 logs, 5·8 logs and r2·6 (Q6·2) logs were found for HIV, BVDV, PSR, SV40 and EMC, respectively. For all tested viruses the precipitation of fraction III from fraction II + III was the most effective step. From the overall reduction factors it appears that cold ethanol fractionation, although capable of reducing viral infectivity to a significant extent, is not sufficient to meet the requirements of regulatory bodies for viral safety of immunoglobulin products. However, pH 4 treatment contributes effectively to the viral safety of the final products. Treatment at pH 4·05 and 37°C for 16 h, as is applied to IVIg, yields reduction factors of r8·4 logs, r4·0 logs, r7·1 logs, 4·8 logs and 1·4 logs for HIV, BVDV, PSR, SV40 and EMC, respectively. The effectiveness of this process step could be enhanced by extending incubation to 40 h at pH 4·25 compared to 16 h at pH 4·05. The extended incubation, as applied in the production of IMIg, yields a reduction of infectivity of SV40 by r5·5 (Q8·0) logs and of EMC by r4·1 (Q7·1) logs. Storage of IMIg, which is formulated as a solution, at 2–8°C also contributes to virus safety. For storage periods of 8 weeks or longer, reduction factors of 2 to 6 logs were found for all viruses, except for BVDV which remained unaffected. These data indicate that the production processes for IVIg and IMIg as described here have sufficient virus reducing capacity to achieve a high margin of virus safety. = 1998 The International Association of Biological Standardization
Introduction Although immunoglobulin products, derived from human plasma by Cohn's cold ethanol fractionation process,1,2 have been regarded as generally safe,3,4 transmission of hepatitis non-A, non-B (NANBH) and hepatitis C by intravenous immunoglobulin (IVIg) has been reported occasionally.5±12 These transmissions were associated with products manufactured by processes that differed in a number of
*To whom correspondence should be addressed: J. Over, Department of Product and Process Development, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. 1045±1056/98/040267 + 10 $30.00/0
aspects, but included in all cases cold ethanol fractionation. This suggests that the Cohn process has only a limited potential to remove or inactivate viral contaminants, if GMP failures are not included as a possible cause of such transmission. Manufacturers of immunoglobulin products have therefore been urged to both validate their production processes for the capability to clear viruses and implement additional virus inactivation steps when no efficacious virus inactivation step appears to be present.13,14 Treatment of intramuscular immunoglobulin products (IMIg) at pH 4, in the presence of a low concentration of pepsin, was originally developed to 7 1998 The International Association of Biological Standardization
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reduce both anti-complementary activity and contaminating enzymes of the contact activation system, in order to render these products suitable for intravenous administration.15 Subsequently, it was shown that such treatment may also improve viral safety of IVIg.16±18 To improve the safety margin of IMIg, we now have introduced pH 4 treatment as a specific virucidal step in the production process of IMIg. This study describes the assessment of the virus reducing capacity of the Cohn fractionation process, as well as that of the pH 4 step in the production processes of both IVIg and IMIg. With respect to low pH treatment, a comparison is made between the virus reducing capacity of incubation at pH 4⋅05 (for 16 h) and pH 4⋅25 (for 30±40 h).
removal of cryoprecipitate removal of prothrombin complex (optional) removal of C1 –esterase inhibitor (optional) Cryo-depleted plasma (8% EtOH, –3 °C, pH 7.4) Suspension I precipitate I Supernatant I (25% EtOH, –5 °C, pH 6.8) Suspension II + III supernatant II + III Precipitate II + III
Suspended precipitate II + III (12% EtOH, –3 °C, pH 5.1) Suspension III precipitate III Supernatant III (25% EtOH, –5 °C, pH 7.3) Suspension II supernatant II
Materials and methods Plasma and ethanol fractionation process Plasma and in-process fractions thereof were derived from the routine production processes for immunoglobulin products of the Sanquin Blood Supply Foundation, CLB division (CLB; formerly known as Central Laboratory of the Netherlands Red Cross Blood Transfusion Service). The starting plasma pool, collected from healthy, nonremunerated volunteer donors, was regularly made up of 5000 donations, which were all tested and found negative for HBsAg and for antibodies against human immunodeficiency virus types 1 and 2 (HIV-1/2), human T-lymphotropic virus types I and II (HTLV-I/II) and hepatitis C virus (HCV). Both IMIg and IVIg products were manufactured from pooled plasma by the cold ethanol fractionation process according to a combination of Oncley's method2 (first part) and that of Kistler and Nitschmann 19 (second part) (Fig. 1). Temperatures were kept within a range of 20⋅5°C in all steps. All precipitates from the ethanol fractionation process were separated by filtration. With the exception of fraction II, filtration of all fractions was facilitated by adding filter aid (Celite; Celite Corp., Denver, CO).20 Virus reduction data for cryoprecipitation and precipitation of Cohn fraction I were kindly made available by the Finnish Red Cross Blood Transfusion Service (FRC; by courtesy of Prof. G. MyllylaÈ). The process conditions for these steps at the FRC were identical to those of the CLB. Moreover, these virus validation studies of the FRC had also been carried out by CLB Virus Safety Services.
albumin
Precipitate II Suspended precipitate II Lyophilized precipitate II Dissolved powder II pH 4.05, 16 h, 37 °C, pepsin
pH 4.25, 40 h, 37 °C, (no pepsin) dia-/ultrafiltration
i.v. immunoglobin
normal i.m. immunoglobulin, hyperimmune immunoglobulins
Figure 1. Flow chart of the plasma fractionation process for immunoglobulin products as applied by CLB.
pH 4 treatment For the production of the IVIg product, fraction II was dissolved in glucose-containing buffer and lyophilized to remove residual ethanol. Following dissolution of the lyophilized powder II in water for injection to a 7±8% (W/W) protein solution, the pH was carefully lowered to 4⋅05 using 0⋅5 m HCl. Following the addition of pepsin (7 mg/l; Fluka, Buchs, Switzerland) the solution was incubated at 37°C for 16 h. Thereafter, the pH was adjusted to 6⋅25. After sterile filtration and filling, the product was freeze-dried. For the production of IMIg, the procedure was identical to that of IVIg, up to the pH 4 step. To
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enhance the virus-reducing capacity of that step, the time of incubation at pH 4⋅05 was prolonged, but this induced significant amounts of aggregates in the product. At pH 4⋅25 however, the immunoglobulin solution could be incubated for up to 40 h at 37°C without significant polymer generation. Therefore, we carried out pH 4 treatment for IMIg at pH 4⋅25 at 37°C for 40 h, in the absence of pepsin. After incubation at pH 4 a diafiltration step was carried out to replace glucose by glycine and to raise the pH to 6⋅85. Thereafter the solution was concentrated to 16% (W/W) protein concentration, filtered sterile and filled in final containers. The final product was stored as a solution at a temperature of 2±8°C. The pH was measured using a meter (WTW, model 501 or 530; Weilheim, Germany) and a combination pH electrode (Russell pH Ltd., Auchtermuchty, U.K.), calibrated daily using calibration buffers of pH 4⋅00 and 7⋅00 at 20°C. In the course of the incubation the pH rose by about 0⋅1 pH units. Down-scaling of manufacturing steps For measurement of the virus-reducing capacity all tested steps were scaled down from production to laboratory scale. To demonstrate that the downscaling of individual steps was valid, process validation was performed to compare parameters such as pH, protein and IgG contents of the fractions obtained during both routine production and the down-scaled process. However, due to the small volumes used in the down-scaled process, it was difficult to apply the same filtration technique for the removal of precipitates as was used in routine production. Therefore, centrifugation instead of filtration was used to recover the precipitates of the down-scaled process. The virus removal effect of both separation techniques was compared in a separate virus validation study with pseudorabies virus (PSR; also known as suid herpesvirus 1) and simian virus 40 (SV40). Virus culturing and infectivity assays To determine the virus-removing or -inactivating capacity of the various process steps, spiking experiments were performed using the lipidenveloped viruses HIV, bovine viral diarrhoea virus (BVDV) and PSR, and the non-lipid-enveloped viruses SV40 and encephalomyocarditis virus (EMC). HIV was studied being a relevant virus,
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whereas BVDV and EMC were tested as specific model viruses for HCV and hepatitis A virus (HAV), respectively. SV40 and PSR were chosen as general model viruses for non-lipid-enveloped and lipidenveloped DNA viruses, respectively. Virus inocula of HIV (strain HTLV-IIIB), BVDV (strain NADL), PSR (strain Bartha K61), SV40 (strain Pa-57) and EMC (strain EMC) were prepared using H9, EBTr, PD5, CV-1 and Vero cells, respectively. MT2 cells were used for HIV cytotoxicity testing and infectivity assays. For the other viruses the cells used for the infectivity assays were identical to those for preparing virus inocula. The virus inocula for spiking of process steps were derived from virus culture supernatants. However, to obtain a high titre HIV inoculum, the culture supernatant was concentrated 100-fold using ultracentrifugation. All starting materials were spiked with maximally 5% (V/V) of inoculum, which was shown not to affect significantly the properties of the respective starting materials. Starting materials of the process steps were examined for cytotoxic effects. Aliquots of serial dilutions of starting material were added to cell cultures and subsequently inspected microscopically for cytotoxic effects. To ensure the accuracy of the infectivity assays, only non-cytotoxic concentrations of starting materials were used. Infectivity of samples was determined using 50% tissue culture infectious dose (TCID50) assays. Fifty ul aliquots (or 0⋅5 ml for HIV) of 12 (or 8 for HIV) 10-fold serial dilutions were tested in quadruplicate in cell culture. Cell cultures which were incubated for 28, 6, 5, 14 or 6 days (HIV, BVDV, PSR, SV40 and EMC, respectively) were inspected for cytopathic effects. For HIV the read-out was confirmed by reverse transcriptase enzyme activity assay.21 To achieve a lower limit of detection of viral infectivity, larger volumes of samples (20 ml at maximum, depending on the cytotoxicity of the sample) were also tested in bulk cultures in duplicate. The detection limit of the TCID50 and bulk culture assays was calculated with the formula: titre [TCID50/ml] = log (1/V) (V, total volume tested; [V] = ml). As the virus validation studies reported here were carried out before 1994, a few guidelines of the Paul Ehrlich Institute relating to virus infectivity assays22 were not met in this study (i.e. 3-fold serial dilutions to be tested in 8-fold, and TCID50 calculation according to the Spearman-KaÈrber formula). As a result of this, the sensitivity of the infectivity assays in this study was about 0⋅5±1 log TCID50 unit instead of R0⋅5 log.
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Table 1. Protein and IgG recovery of Cohn fractionation steps during down-scaled processing and in routine production Down-scaled processing (n = 3) Plasma fraction
Large-scale production (n = 5)
Protein (%) (mean 2 SD)
IgG (%) (mean 2 SD)
Protein (%) (mean 2 SD)
IgG (%) (mean 2 SD)
100 (55 2 4 g/l) 91 2 1 67 2 3 21 2 7 11 2 1 11 2 1
100 (8⋅2 2 0⋅6 g/l) 101 2 11 7 2 1* 88 2 9 70 2 4 18 2 0
100 (56 2 14 g/l) 89 2 9 71 2 11 23 2 7 11 2 2 11 2 3
100 (6⋅4 2 0⋅2 g/l) 89 2 5 3 2 1* 91 2 7 65 2 9 21 2 3
Cryo-depleted plasma Supernatant I Supernatant II + III Precipitate II + III Supernatant III Precipitate III
*Difference statistically significant, P Q 0·001.
Results Experimental design and down-scaling of the IVIg and IMIg production process In this study, down-scaling experiments were performed to make the scale of the virus validation studies representative for the routine production scale. The scale reduction, expressed as percentage of the production scale, varied between 0⋅001% and 0⋅03% dependent on the step tested. Comparing the recovery of total protein and immunoglobulin G (IgG) in various fractionation steps, we did not detect significant differences between the downscaled processing and large-scale production, except for the residual IgG content in the supernatant of
fraction II + III (Table 1). As both the conditions and the performance of the process at small laboratory scale were similar to those at full production scale, the virus reductions found are most likely to be representative for the virus reduction in routine production. When centrifugation of precipitates of the cold ethanol fractionation was compared to filtration with respect to removal of PSR and SV40, no significant differences were found between the two separation techniques for both fraction II + III and fraction III (Table 2). Therefore, the centrifugation in the virus validation experiments is considered to be a representative alternative to the filtration technique used in the large-scale production setting.
Table 2. Viral reduction factors (in log TCID50 units) for PSR and SV40 for the precipitation of fractions II + III and III: comparison between centrifugation and filtration Centrifugation
Fraction II + III: Suspension II + III² Supernatant II + III Precipitate II + III Fraction III: Suspension III² Supernatant III Precipitate III
Filtration
PSR*
SV40*
PSR
SV40
−0⋅6/−0⋅1 2⋅6/r3⋅4 −0⋅3/0⋅2
−0⋅4/−0⋅8 r3⋅4/r2⋅9 0⋅5/−0⋅2
−0⋅1 r3⋅4 −0⋅1
−0⋅8 r3⋅2 −0⋅1
0⋅2/0⋅5 3⋅3/r3⋅9 0⋅8/1⋅0
−0⋅4/0⋅3 2⋅4/2⋅9 −0⋅2/0⋅2
0⋅5 3⋅2 0⋅5
0⋅3 2⋅6 −0⋅1
*The results of two experiments are shown. †Suspensions II + III and III were incubated for 1 h and 2 h respectively, before separation of the precipitates.
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Table 3. Viral reduction factors (in log TCID50 units) of the Cohn fractionation process for fraction II Process step Removal of cryoprecipitate
HIV
BVDV
PSR
SV40
EMC
0⋅2
−0⋅4*
0⋅4
1⋅4
0⋅3*
Fraction I: Suspension I* (1 h/10 h)² Supernatant I* (1 h/10 h) Precipitate I* (1 h/10 h)
1⋅0/ND 0⋅8/0⋅8 1⋅4/1⋅4
0⋅2/ND 0⋅4/0⋅7 0⋅5/0⋅8
0⋅5/ND r2⋅4/1⋅3 −0⋅7/−0⋅1
0⋅8/ND 2⋅0/2⋅6 0⋅9/0⋅4
0⋅3/ND 0⋅0/−0⋅2 0⋅9/1⋅2
Fraction II + III: Suspension II + III (1 h/15 h)² Supernatant II + III (1 h/15 h) Precipitate II + III (1 h/15 h)
0⋅9/ND 1⋅1/2⋅9 R0/R0
0⋅2*/ND 0⋅9*/ND −0⋅2*/ND
−0⋅6/ND 2⋅6/2⋅6 −0⋅3/−0⋅5
−0⋅4/ND r3⋅4/r3⋅4 0⋅5/0⋅3
ND/ND ND/ND ND/ND
Fraction III: Suspension III (2 h/15 h)² Supernatant III (2 h/15 h) Precipitate III (2 h/15 h)
0⋅2/−0⋅1 3⋅5/3⋅0 −1⋅0/ND
0⋅2/0⋅5 r2⋅6/r2⋅6³ 0⋅5/ND
0⋅2/−0⋅1 3⋅3/3⋅5 0⋅8/ND
−0⋅4/−0⋅7 2⋅4/3⋅0 −0⋅2/ND
0⋅2/0⋅2 r2⋅6§/r2⋅8§ −0⋅1/0⋅2
3⋅0
r2⋅6³
4⋅6
5⋅8
r2⋅6§
Overall reduction factor¶
ND: not determined. *Results of virus validation studies carried out by CLB Virus Safety Services on behalf of the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland); process conditions were identical to those of CLB. ²Duration of incubation after reaching the final process conditions for that step and before separation of the precipitate was carried out. One hour and 2 h incubation time, respectively, are the routine conditions. ³ Q 5⋅5; § Q 6⋅2. ¶In accordance with EC guideline EC/8115/89-EN,13 only reduction factors larger than 1⋅0 log (printed in bold) were used to calculate the overall reduction factors.
Virus reduction in the cold ethanol fractionation process The virus-spiking studies of the cold ethanol fractionation process steps leading to fraction II show that for suspensions I, II + III and III no significant reduction of viral infectivity was found in spite of the presence of ethanol concentrations up to 25%, regardless of whether a short (1±2 h) or long (10±15 h) incubation time was applied after achieving the process conditions for inducing the precipitate (Table 3). After separation of the precipitates essentially all virus was recovered in the precipitate. This resulted in some reduction of infectious virus in the corresponding supernatant, supernatant I showing the smallest reduction and supernatant III the largest (Table 3). Therefore, the mechanism of viral reduction in supernatant III is removal rather than inactivation. Virus inactivation by low pH treatment The reduction of virus infectivity in the IVIg preparations treated at pH 4⋅05 or pH 4⋅25 in the presence of pepsin demonstrates that low pH treatment has a significant virus inactivating
capacity (Table 4). Both at pH 4⋅05 and at pH 4⋅25 HIV and PSR were reduced within 16 h to titres below the detection limit. However, BVDV was not reduced below the detectable titer until treated for 30 h at pH 4⋅25 (infectivity still detectable after 16 h when assayed in a bulk culture). The kinetics of virus inactivation at pH 4⋅05 (Fig. 2) show that lipid-enveloped viruses were inactivated more efficiently than non-lipid-enveloped viruses. None of the tested non-lipid-enveloped viruses were inactivated to an undetectable titre within 40 h of treatment at pH 4⋅25 (Table 4). However, the kinetics of inactivation of EMC show that a long incubation period at pH 4⋅25 is more effective than a relatively short incubation at pH 4⋅05 (Fig. 3). This is a result of the duration of incubation rather than to the small difference in pH, as the kinetics of inactivation at both pH values are similar. Reduction of viral infectivity by pH 4⋅25 treatment of IMIg preparations in the absence of pepsin was equivalent to that of IVIg in the presence of pepsin (Table 4). This is also clearly shown by the kinetics of inactivation of EMC (Fig. 3). Thus it seems that the presence of trace amounts of pepsin
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Table 4. Viral reduction factors (in log TCID50 units) of incubation at pH 4⋅05 and pH 4⋅25 for intravenous immunoglobulin preparations (in the presence of pepsin) and intramuscular preparations (in the absence of pepsin) HIV
BVDV
PSR
SV40
EMC
Intravenous immunoglobulin Incubation for 16 h at pH 4⋅05 Incubation for 16 h at pH 4⋅25 Incubation for 40 h at pH 4⋅25
r8⋅4 r7⋅3 r7⋅3
r4⋅0* r3⋅5² r6⋅0
r7⋅1 r8⋅0 r8⋅0
4⋅8 r5.5³ r5⋅5³
1⋅4 2 0⋅3 2⋅6 r4⋅1§
Intramuscular immunoglobulin Incubation for 16 h at pH 4⋅25 Incubation for 40 h at pH 4⋅25
r6⋅8 r6⋅4
r4⋅0* r6⋅5
r6⋅0 q8⋅5
r5⋅4¶ r5⋅4¶
2⋅6 r3⋅6>
* Q 6·5; †Q6·0; ‡Q8·0; §Q7·1; ¶Q7·9; >Q7·2.
(in the case of IVIg) did not contribute significantly to virus reduction during incubation at pH 4⋅25. Virus inactivation during storage Storage of IMIg (which is formulated as a solution) at 2±8°C revealed some virus inactivating potential (Table 5). The extent of inactivation varied with the virus tested, HIV being affected the most, while BVDV showed little or no inactivation at all. It should be emphasized that the presence of thiomersal as an antimicrobial additive to the 15-ml filling of IMIg resulted in the demonstration of somewhat lower reduction factors due to its cytotoxicity in the titration assay: viral infectivity could be assayed only at relatively high test sample dilutions. Therefore, the real reduction by storage in the presence of thiomersal was likely to be similar to that in the absence of thiomersal.
The total reduction factors for the entire production processes for both types of immunoglobulin products are summarized in Table 6. Discussion When an overall capacity of at least 6 logs reduction of model viruses is applied as criterion for processes yielding virus safe products, the data show that the version of Cohn's cold ethanol fractionation process, as is in use at CLB, is unable to ensure virus safety of the immunoglobulin products on its own (Table 3). Precipitation of fraction III is the only process step in which viruses were consistently removed to a significant extent. As a reduction of only about 3 logs was achieved for all tested viruses, this process step cannot be regarded as an effective virus reducing step, for which a reduction of at least 4 logs holds.23 For suspensions I, II + III and III no
9 8
8 EMC
7
6 SV40
5 4
log TCID50
log TCID50
7
BVDV
3
PSR
2
5
pH 4.25 – pepsin
4 3
pH 4.25 + pepsin
2
HIV
1
pH 4.05 + pepsin
6
1 0
5
10 Time (h)
15
20
Figure 2. Kinetics of virus inactivation in IVIg solutions at pH 4⋅05 (pepsin present). Open symbols indicate infectivity titers below the detection limit.
0
5
10
15
20 25 Time (h)
30
35
40
45
Figure 3. Kinetics of inactivation of EMC at three regimes of low pH treatment.
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Table 5. Viral reduction factors (in log TCID50 units) of storage conditions for intramuscular immunoglobulin preparations Storage time
HIV
BVDV
PSR
SV40
EMC
With thiomersal:* 8 weeks 12 weeks 16 weeks
r3⋅7 r3⋅7 r3⋅7
1⋅0/0⋅4 1⋅5/0⋅2 ND/0⋅9
r2⋅3 r2⋅5 r6⋅2
r1⋅6 r1⋅6 r1⋅6²
r2⋅3 r2⋅3 r2⋅3³
Without thiomersal:* 8 weeks 12 weeks 16 weeks
4⋅7 r5⋅3 r6⋅0§
r2⋅7 r3⋅2 r5⋅0¶
r4⋅3 r4⋅3 r4⋅3>
2⋅9 2⋅9 r3⋅4**
1⋅2 0⋅8 0⋅5
ND: not determined. *Thiomersal is added as an anti-microbial agent to the 15 ml multi-dose filling, but not to the 1, 2 and 5 ml fillings of the IMIg products of CLB. ² Q 5⋅1; ³ Q 5⋅8; § Q 6⋅6; ¶ Q 7⋅4; > Q 6⋅7; ** Q 7⋅1.
virus reduction at all (i.e. less than 1 log TCID50) was found. This means that none of the tested viruses were inactivated by ethanol concentrations up to 25% at a temperature of −3°C or −5°C. The results further show that the viruses preferentially go with the precipitates, as has also been found earlier.24±29 The limited virus reducing capacity of the cold ethanol fractionation found in this study is consistent with other reports,26±32 although in more early work a higher virus reducing capacity has been reported, including inactivation by ethanol, e.g. for HIV.24,25,33,34 Part of this discrepancy may be explained by not strictly applying the low temperatures of the cold ethanol fractionation process in the laboratory experiments, as HIV is sensitive to ethanol at temperatures above zero.31 Not all cold fractionation process steps have been validated in
this study, so the actual virus reduction may be larger than that demonstrated in this study. Still, a specific virucidal step seems necessary to ensure viral safety of the immunoglobulin products. A similar conclusion has been drawn by others,7,9,26,28,29,35 some of them referring to the NANBH transmissions reported in the literature. Treatment at pH 4 appears to be an effective virucidal step, as reduction factors for the tested viruses are about 4 logs or higher (Table 4). This finding is in accordance with results published previously.3,16±18 The lack of a synergistic effect exerted by the presence of trace amounts of pepsin is in contrast to previous studies,17,18 where it was shown that pepsin enhances the overall effectiveness of the pH 4 treatment. Part of the discrepancy may be due to the fact that both reported studies
Table 6. Overall viral reduction factors (in log TCID50 units) for intravenous and intramuscular immunoglobulins Process step
HIV
BVDV
PSR
I.v. immunoglobulin Cohn fractionation (Table 3) Incubation for 16 h at pH 4⋅05 (Table 4) Overall reduction factor:
3⋅0 r8⋅4 r11⋅4
r2⋅6* r4⋅0³ r6⋅6
I.m. immunoglobulin Cohn fractionation (Table 3) Incubation for 40 h at pH 4⋅25 (Table 4) Storage for q8 weeks (Table 5) Overall reduction factor:
3⋅0 r6⋅4 4⋅7 r14⋅1
r2⋅6* r6⋅5 Q1⋅0 r9⋅1
* Q 5⋅5; ² Q 6⋅2; ³ Q 6⋅5; § Q 7⋅9; ¶ Q 7⋅2.
SV40
EMC
4⋅6 r7⋅1 r11⋅7
5⋅8 4⋅8 10⋅6
r2⋅6² 1⋅4 r4⋅0
4⋅6 q8⋅5 r2⋅7 q15⋅8
5⋅8 r5⋅4§ r4⋅3 r15⋅5
r2⋅6² r3⋅6¶ 2⋅9 r9⋅1
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were performed at pH 4⋅0 and pH 4⋅05, respectively, while we applied pH 4⋅25. Kempf et al. 18 showed that the synergistic effect of pepsin increases with decreasing pH. Another explanation may be that the model viruses used in the reported studies were not the same as used here. Kempf et al. demonstrated this effect only for the relatively resistant vesicular stomatitis virus (VSV), but not for four other lipid-enveloped viruses tested. In the study of HaÈmaÈlaÈinen et al. 17 Semliki forest virus proved to be sensitive to pepsin, while VSV was rather insensitive. Immunoglobulin for intramuscular administration which has not been treated at pH 4 has never been implicated in transmission of HIV, NANBV, HCV or hepatitis B virus, despite decades of routine clinical use. Although not all production steps possibly contributing to virus inactivation or removal (e.g. freeze-thawing of the precipitates or freeze-drying3,25,29) have been validated in this study, the good clinical record of viral safety of IMIg preparations especially indicates that also factors other than the Cohn fractionation steps must play a role in achieving viral safety. It should be taken into account that virus safety of immunoglobulin products with respect to some relevant viruses, like HAV and parvovirus B19, is most probably increased by immune neutralization due to the presence of significant amounts of antibodies neutralizing HAV and parvovirus B19. Furthermore, the viral safety of intramuscular preparations may be positively influenced by storage of the final product in its liquid form. This study shows that the storage conditions of IMIg preparations at (near-)neutral pH has a virus inactivating effect, although the extent of inactivation depended on the virus tested. This effect of storage has also been described by other groups.3,17,32 Also, other factors contribute to reducing the viral transmission risk, such as the use of plasma from non-remunerated donors, the policy to discourage persons who carry an increased risk for blood-borne viruses (like intravenous drug abusers) to donate, and the screening of all donations for viral markers like anti-HIV-1/2, anti-HCV and HBsAg. Over the years, viral transmission caused by the use of CLB's IVIg and IMIg products has never been reported to us, whereas transmission of NANBH through IVIg products has occurred incidentally elsewhere.5±12 Several possible reasons have been suggested to explain these transmissions. Among these are production in a pilot plant, an excessive viral load in the starting plasma, or GMP failure.7,10
It now turns out that the Cohn fractionation process on its own is insufficient to guarantee virus safety. This is also demonstrated by more recent transmissions of HCV by an intravenous immunoglobulin product which was prepared by Cohn fractionation, but without additional viral reduction methods like organic solvent/detergent treatment or low pH.11 However, transmission of HCV by this product only occurred after the introduction of anti-HCV screening of the plasma donations by second-generation assays,4,12 which are more efficient in detecting antibodies to HCV than the first-generation assays (anti-C100, anti-C33c and anti-core instead of anti-C100 only in the case of the latter36). Most likely, antibodies to HCV originally present in the plasma pool contributed to the viral safety of the product, possibly by formation of complexes with the virus which were diverted away from the final immunoglobulin product in the purification process, while uncomplexed virus is not. This hypothesis was already postulated in 199037 and has led to a study in chimpanzees which showed no risk of transmitting HCV by immunoglobulin products derived from plasma screened for anti-HCV by first-generation assays.38 The transmission of NANBV by a product, prepared by the Cohn fractionation process and treated at pH 4⋅05 for 16 h10 cannot be explained in this way. An exceptionally high viral load of the starting plasma pool or a GMP failure have been postulated as possible causes.10 It should be noted that in this case only one product batch was involved, which later turned out to be negative for HCV-RNA by PCR testing.39 Reconsidering all data available, it may be concluded that extended incubation at pH 4 is capable of effectively inactivating lipid-enveloped viruses. In combination with the limited virus reducing capacity of the Cohn fractionation, pH 4 treatment reduces the risk of transmission of lipid-enveloped viruses by the IVIg product to a minimum. The infectivity of non-lipid-enveloped model viruses is less readily reduced below the detection limit by low pH treatment, which urges prolonged treatment (up to 40 h) at low pH. With respect to relevant non-enveloped viruses, immune neutralization also contributes to viral safety, while for IMIg some additional virus reduction is obtained by the storage condition of the liquid final product. The latest safety measure that has now become possible is screening of the starting plasma for the presence of viral DNA or RNA of relevant viruses by the PCR technique,40,41 as has been in use in our
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centre for HCV since late 1995. As a result of all these measures IVIg and IMIg products are obtained which can be considered to carry a minimal risk of virus transmission. Acknowledgements We thank Prof. G. MyllylaÈ of the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland, for making available part of the virus validation data of the cryoprecipitation and Cohn fractionation processes. Prof. W. G. van Aken (CLB) is gratefully acknowledged for his constructive criticism on this paper. References 1. Cohn EJ, Strong LE, Hughes WL Jr et al. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946; 68: 459±475. 2. Oncley JL, Melin M, Richert DA et al. The separation of the antibodies, isoagglutinins, prothrombin, plasminogen and b1-lipoprotein into subfractions of human plasma. J Am Chem Soc 1949; 71: 541±550. 3. Cuthbertson B, Perry RJ, Foster PR et al. The viral safety of intravenous immunoglobulin. J Infection 1987; 15: 125±133. 4. Yap PL. The viral safety of intravenous immune globulin. Clin Exp Immunol 1996; 104 (Suppl 1): 35±42. 5. Lane RS. Non-A, non-B hepatitis from intravenous immunoglobulin. Lancet 1983; ii: 974±975. 6. Lever AML, Webster ADB, Brown D et al. Non-A, non-B hepatitis occurring in agammaglobulinaemic patients after intravenous immunoglobulin. Lancet 1984; ii: 1062±1064. 7. Ochs HD, Fischer SH, Virant FS et al. Non-A, non-B hepatitis and intravenous immunoglobulin. Lancet 1985; i: 404±405. 8. Weiland O, Mattson L, Glaumann H. Non-A, non-B hepatitis after intravenous gammaglobulin. Lancet 1986; i: 976±977. 9. BjoÈrkander J, Cunningham-Rundles C, Lundin P et al. Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinemia or IgG subclass deficiency. Am J Med 1988; 84: 107±111. 10. Williams PE, Yap PL, Gillon J et al. Transmission of non-A, non-B hepatitis by pH 4-treated intravenous immunoglobulin. Vox Sang 1989; 57: 15±18. 11. Anonymous. Outbreak of hepatitis C associated with intravenous immunoglobulin administrationÐUnited States, October 1993±June 1994. Morb Mort Wkly Report 1994; 43: 505±509. 12. Yu MW, Mason BL, Guo ZP et al. Hepatitis C transmission associated with intravenous immunoglobulins. Lancet 1995; i: 1173±1174. 13. EEC Regulatory Document. Note for Guidance: Validation of virus removal and inactivation procedures. Biologicals 1991; 19: 247±251.
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Received for publication 8 April 1998; accepted 20 July 1998