SEARCH

SEARCH BY CITATION

Keywords:

  • Flebogamma;
  • intravenous immunoglobulin;
  • pathogen safety;
  • nanofiltration

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Flebogamma® 5% dual inactivation and filtration (DIF), a new 5% liquid intravenous immunoglobulin with a stability of 2 years when stored at temperatures between 2 and 30°C, has been developed. This new product is the result of the accumulated experience provided by Flebogamma®, with more than 30 million grams administered since 1992 in Europe and the United States, and the implementation of the latest technology to improve Flebogamma® even more by increasing its viral safety margin further. In addition to the specific inactivation stage for Flebogamma® 5% (pasteurization), the new process includes a solvent–detergent treatment and nanofiltration through a Planova filter down to 20 nm. The preparation presents a mean purity of 99·6 ± 0·2% with a correct chromatographic profile. Percentage values of immunoglobulin (Ig)G subclasses are equivalent to the physiological values of normal serum. The content in IgA as well as other possible impurities is very low, and the product presents a mean result of 109 ± 5% in the Fc fragment functionality assay, demonstrating the integrity of the IgG molecule. The functionality is also reflected in neutralization tests carried out against poliomyelitis, diphtheria, measles and vaccinia which, apart from the antibody titres determined by enzyme-linked immunosorbent assay, guarantees that antibodies are capable of reacting against these pathogens. Regarding safety, the combination of multiple methods with capacity to inactivate or remove biological agents which include chemical inactivation, heat inactivation, nanofiltration and precipitations, with very different mechanisms of action, provides Flebogamma® 5% DIF very wide margins of safety regarding to potential pathogens.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Flebogamma® 5% dual inactivation and filtration development (DIF) is a new, sterile, ready-to-use, liquid intravenous immunoglobulin (IVIG) stabilized with 5% sorbitol, stable at room temperature (2–30°C) for 2 years. One of the main objectives during the design of Flebogamma® 5% DIF was to preserve the excellent characteristics of Flebogamma® 5% liquid IVIG, which has been marketed by Instituto Grifols (Barcelona, Spain) for more than 15 years. Flebogamma® 5% has shown very good results in efficacy and tolerability, as more than 30 million grams of Flebogamma® have been used globally. It is marketed in more than 30 countries, including Europe and the United States.

The project for the development of Flebogamma® 5% DIF began in 1996 with the intention of maintaining the qualities of Flebogamma® and applying the latest-generation technology to achieve very low impurity levels, especially immunoglobulin (Ig)A, as well as to increase its safety margin, apart from more dosing flexibility by making an additional 20 g presentation available.

The process for immunoglobulin purification in Flebogamma® 5% DIF, just as the process for Flebogamma®, is based on Cohn's method [1,2] (cold ethanol fractionation) from normal human plasma. This is followed by stages of sequential precipitation with polyethylene glycol (PEG) and ion exchange chromatography, where impurities are eliminated (see Fig. 1).

image

Figure 1. Summary of Flebogamma® 5% dual inactivation and filtration (DIF) manufacturing process showing the steps validated for inactivation or removal of viruses.

Download figure to PowerPoint

When medicinal products are obtained from biological sources, the risk related to transmissible agents cannot be excluded totally. However, in the case of plasma derivatives the risk is reduced by selection of donors, screening of individual donations and plasma pools, together with pathogen elimination steps (through inactivation or removal mechanisms) included in the production process. These steps were validated using relevant model viruses for Flebogamma® 5% DIF. Figure 1 shows the production process, including the steps validated for inactivation/removal of viruses.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Characterization studies

Flebogamma® 5% DIF lots (n = 9–29) were studied. The following tests were performed: purity cellulose acetate membrane electrophoresis (CAME) [3]; protein identification (immunoelectrophoresis) [4]; molecular distribution [high performance liquid chromatography (HPLC)][5]; protein concentration (Bradford) [6]; determination of IgG subclasses and possible accompanying proteins (IgA, IgM, albumin, transferrin) by immunonephelometry; Fc fragment integrity (haemolysis) [7]; anti-complementary activity (ACA, haemolysis) [8]; prekallikrein activator (PKA, chromogenic assay) [9]; anti-A, anti-B, anti-D haemagglutinins (agglutination) [10,11]; and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). A broad spectrum of antibody titres was determined by enzyme-linked immunosorbent assay (ELISA) or neutralization assay [12–14].

Viral safety studies

Down-scaled laboratory experiments, validated for equivalence to the industrial-scale process, were performed. Agents of different physicochemical properties were spiked into production process materials, among them models for human immunodeficiency virus (HIV), herpesvirus, hepatitis B, hepatitis C, West Nile virus (WNV), hepatitis A (HAV) and B19 virus. A volume of each agent was spiked in at least two independent experiments for each studied step and agent. The titres of initial load and processed samples were determined by infectivity assays. The ability of a process to eliminate viruses is shown by means of the reduction factor (RF, log10), which compare the virus titres before and after the step. All studies were performed and designed according to the relevant International Guidelines [15–17].

Nanofiltration through Planova 20N filters (20 nm; Asahi Kasei, Japan) was chosen after a filtration robustness assessment study. A comparison of two 20 nm nanofilters (Planova 20N and ‘filter A’) was performed by conducting experiments with porcine parvovirus (PPV, 18–26 nm, among the smallest viruses known) spiked in a solution of a plasma protein.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Product characterization

Cellulose acetate electrophoresis demonstrate that Flebogamma® 5% DIF is a high-purity product, with a purity of 99·6 ± 0·2% (n = 19).

During the determination of impurities using immunoelectrophoresis against whole human anti-serum (Fig. 2), the only visible precipititation arc corresponds to IgG and the presence of no other plasma protein is detected.

image

Figure 2. Immunoelectrophoresis of three lots of Flebogamma® dual inactivation and filtration (DIF) against total human anti-serum. From left to right, 1: Pool normal human plasma; 2: Flebogamma® 5% dual inactivation and filtration (DIF) lot 1; 3: Flebogamma® 5% DIF lot 2; 4: Flebogamma® 5% DIF lot 3; 5: pool normal human plasma.

Download figure to PowerPoint

A more exhaustive analysis of possible impurities was performed using immunonephelometric techniques (Table 1) and resulted in undetectable or practically undetectable levels.

Table 1.  Concentration range of impurities detected in Flebogamma® 5% dual inactivation and filtration (DIF) (n = 19).
ImpuritiesRange mg/ml
  1. Ig: immunglobulin.

IgA< 0.003–0.013
IgM< 0.002
Transferrin< 0.002–0.009
Albumin< 0.002–0.007

IgA levels in Flebogamma® 5% DIF are practically undetectable. It is known that the IgA content in IVIG concentrates is important, as there is a possibility that IgA-deficient patients who have developed IgA antibodies may present anaphylactic reactions during treatment with IVIG if the IgA is present in high concentrations.

IgG may present different molecular forms, including fragments, monomer, dimer and polymer and/or aggregates. It has been reported that the polymers and/or aggregates may activate the complement in the absence of antigen, increasing the possibility of anaphylactic reactions [18]. The presence of fractions indicates degradation of the molecule [18]. HPLC was used to determine the molecular distribution of the IgG present in Flebogamma® 5% DIF (Table 2), with a high monomer and dimer content and undetectable levels of polymer/aggregates and fractions.

Table 2.  Molecular distribution in Flebogamma® 5% dual inactivation and filtration (DIF) (n = 19).
 Molecular distribution
Monomer + dimer (%)99·8–100
Polymer/aggregates (%)≤ 0·2
Fractions (%)≤ 0·3

The concentration of IgG subclasses was determined using nephelometry, and the mean results are shown in Fig. 3. The percentage of IgG subclasses may be considered similar to normal serum values [19].

image

Figure 3. Immunoglobulin (Ig)G distribution among the different subclasses (n = 19).

Download figure to PowerPoint

The functionality of the gammaglobulin was determined by analysing the integrity of the Fc fragment. A study was made of the response of IVIG to tanned red blood cells with rubella antigen to determine the functionality of the Fc fragment [7].

The mean resulting integrity of the Fc fragment of Flebogamma® 5% DIF was 109 ± 5% (n = 19) compared to a biological reference preparation (BRP), therefore proving the functionality of Flebogamma® 5% DIF.

The approximate molecular weights were determined using SDS-PAGE and resulted in a band of approximately 150 kDa under non-reducing conditions; this band corresponds to whole gammaglobulin molecules. Under reducing conditions, disulphide bridges are broken down and there are two bands of molecular weights of about 58 and 25 kDa, corresponding to the heavy and light chain, respectively.

The content in specific antibodies against a broad spectrum of possible pathogens is shown in Table 3. ELISA techniques and neutralization assays were used in the case of poliovirus, measles, diphtheria and vaccinia.

Table 3.  Antibody titres against different pathogens of Flebogamma® 5% dual inactivation and filtration (DIF) n = 9 for vaccinia virus and n = 29 for the remaining pathogens).
PathogenAntibody titres
  1. Ig: immunoglobulin.

Hepatitis B37 ± 24 IU/g Ig
Hepatitis A21 ± 4 IU/ml
Tetanus16 ± 3 IU/ml
Cytomegalovirus26 ± 5 UPEI/ml
Rubella347 ± 54 IU/ml
Varicella8 ± 0·9 IU/ml
Streptococcus pneumoniae285 ± 22 mg/l
Haemophilus influenzae type b15 ± 1 mg/l
Poliovirus type 10·39 ± 0·13 ratio sample/reference preparation
Measles0·50 ± 0·07 ratio sample/reference preparation
Vaccinia20·7 ± 7·2 IU/ml
Diphtheria6 ± 1 U/ml

The product characterization was completed by determining the PKA content, ACA, anti-A and anti-B haemagglutinins and anti-D antibodies, as well as other parameters (see Table 4). In all cases, the parameters meet the requirements of the European Pharmacopoeia.

Table 4.  Other parameters characterized of Flebogamma® 5% dual inactivation and filtration (DIF) (n = 19).
ParameterResults
  1. ACA: anti-complementary activity; PKA: prekallikrein activator; Ig: immunoglobulin.

PKA (IU/ml)< 2
ACA (CH50/mg Ig)0·4 ± 0·1
Anti-A haemagglutininsNo agglutination Dil 1/4–1/32
Anti-B haemagglutininsNo agglutination Dil 1/4–1/16
Irregular antibodiesNo agglutination Dil 1/2
Anti-D antibodiesPass test
Osmolality (mOsm/kg)329 ± 6
pH5·6 ± 0·1
Total protein (mg/ml)48 ± 1
Heat stabilityDoes not show any visible sign of gelation
PyrogensPass test
SterilityNo microbiological growth
Abnormal toxicityNo indication of toxicity

The stability study evaluated 40 batches at temperatures of 5 ± 3°C, 30 ± 2°C and 40 ± 2°C during a maximum period of 30 months. The results show that the product remains stable and maintains its characteristics during 24 months at a temperature between 2 and 30°C.

Viral safety studies

Figure 1 shows the production process of Flebogamma® 5% DIF, including the steps validated for inactivation/removal of viruses. The Flebogamma® 5% DIF production process includes three specific virus elimination steps (labelled green in Fig. 1): pasteurization (60°C, 10 h), solvent–detergent (S/D) treatment and Planova nanofiltration (down to 20 nm). Other steps (labelled purple in Fig. 1) – fraction I and polyethylene glycol (PEG) precipitations, fraction II + III incubation in the presence of ethanol and acid pH treatment – were also studied for their contribution to the product safety.

All viruses tested were inactivated and/or removed effectively (4 to =6 log10 reduction) at each of the following steps: PEG precipitation, pasteurization, S/D treatment and Planova nanofiltration (down to 20 nm).

Process steps related to Cohn fractionation showed lower reduction factors and the acid pH treatment showed high variability of results, depending on the specific pathogen model analysed (range 1·3 to =5·3 log10).

Results of the comparison between Planova 20N and an alternative 20 nm nanofilter (filter ‘A’) are shown in Table 5. PPV removal results obtained with Planova 20 N nanofilters, demonstrated clearly in the presence of the plasma protein (4·37 log10), are similar to those obtained with Flebogamma® 5% DIF (removal of 4·61 log10, data on file). Surprisingly, filter ‘A’ failed to show any significant PPV reduction using that plasma protein. This proves that not all 20 nm nanofilters work with identical efficiency. Planova 20N is more reliable and robust, as the PPV removal results obtained are similar regardless of the protein solution used.

Table 5.  Comparison of two 20 nm nanofilters. Porcine parvovirus (PPV) reduction factors (log10).
Filter ‘A’Planova 20N
Laboratory ALaboratory B
Run 1Run 2Run 3Run 1Run 2Run 3Run 1Run 2Run 3
1·301·130·651·040·651·004·453·864·79
Mean: 0·96   Mean: 4·37

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Flebogamma® 5% DIF is a new unmodified intravenous immunoglobulin solution with the same efficacy and tolerability as Flebogamma®, but with practically undetectable impurity content, increased safety margin and more dosing flexibility.

A characterization study of the product indicates that the immunoglobulin content of this product is 99·6%, and IgG subclasses are distributed in a physiological pattern. A broad spectrum of functional antibodies against various infectious agents is detectable. The IgA content and anti-complementary activity are very low, as are all other contaminants studied.

Stability studies carried out on Flebogamma® 5% DIF guarantee a stability of up to 24 months at room temperature (between 2 and 30°C).

The combination of multiple methods with the capacity to inactivate or remove biological agents, which include chemical inactivation, heat inactivation, Planova nanofiltration (down to 20 nm) and precipitations with very different mechanisms of action, provides the production process of Flebogamma® 5% DIF with very wide margins of safety with regard to potential pathogens. Additionally, the wide spectrum of model agents with different physicochemical properties used provides a high degree of confidence against potentially new emerging agents.

Planova 20N nanofiltration is a robust step because the removal results obtained are similar, regardless of the plasma protein used. Our results suggest that small virus removal capacity may show different levels of robustness, depending upon the type of nanofilter employed.

Grifols has designed and built a new production plant in Barcelona devoted to Flebogamma® 5% DIF production; the result is a new state-of-the-art production plant of approximately 7500 m2 with a processing capacity for 3 million litres of plasma. A twin production plant is under construction in the United States.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The author acknowledges the help in preparing the manuscript from Drs Francisco Belda, Rodrigo Gajardo, Maite López and Núria Marzo, from the Research and Development Area of Instituto Grífols SA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  • 1
    Cohn EJ, Strong LE, Hughes WL 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; 68:45975.
  • 2
    Cohn EJ, Gurd FRN, Surgenor DM, et al. A system for the separation of the components of human blood: quantitative procedures for the separation of the protein components of human plasma. J Amer Chem Society 1950; 72: 46574.
  • 3
    Electrophoresis. European Pharmacopoeia: 2.2.31.
  • 4
    Immunochemical methods. European Pharmacopoeia: 2.7.1.
  • 5
    Liquid chromatography. European Pharmacopoeia: 2.2.29.
  • 6
    Bradford M. Anal Biochem 1976; 72:248.
  • 7
    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Test for Fc function of immunoglobulin. European Pharmacopoeia: 2.7.9.
  • 8
    Test of anticomplementary activity of immunoglobulin. European Pharmacopoeia: 2.6.17.
  • 9
    Prekallikrein activator. European Pharmacopoeia: 2.6.15.
  • 10
    Anti-A and anti-B haemagglutinins (indirect method). European Pharmacopoeia: 2.6.20.
  • 11
    Anti-D antibodies. European Pharmacopoeia: 2.6.26.
  • 12
    Lennette E, Schmidt NJ. Diagnostic procedures for viral, rickettsial and chlamydial infections, 5th edn. Washington, DC: American Public Health Association, 1979.
  • 13
    Rose NE, Hamilton RG, Detrick B (Eds). Manual of clinical laboratory immunology. Washington DC, American Society for Microbiology Press. 1997. p. 690.
  • 14
    Dumas J, Bordet P, Laporte R, Pichon J, Prevot AR. Bacteriologíe medicale. Paris: Ed Medicales Flammarion, 1951.
  • 15
    Revised CPMP Guideline in Virus validation studies. CPMP/BWP/268/95. 1995.
  • 16
    Note for guidance on plasma-derived medicinal products. CPMP/BWP/269/95. 1995.
  • 17
    Quality of biotechnological products viral safety evaluation by biotechnology products derived from cell lines of human or animal origin. CPMP/ICH/295/95. 1995.
  • 18
    Yap PL. Aplicaciones clínicas del tratamiento con inmunoglobulinas intravenosas, Barcelona. JR Prous, 1993.
  • 19
    Vlug A, Nieuwenhuys EJ, Van Eijk RV, Geertzen HG, Van Houte AJ. Nephelometric measurements of human IgG subclasses and their reference ranges. Ann Biol Clin 1994; 52:5617.