Flebogamma® 5% DIF development: rationale for a new option in intravenous immunoglobulin therapy

Abstract

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

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).

Fig. 1.

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

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

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

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.

Fig. 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.

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).

Impurities	Range mg/ml
IgA	< 0.003–0.013
IgM	< 0.002
Transferrin	< 0.002–0.009
Albumin	< 0.002–0.007

Ig: immunglobulin.

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].

Fig. 3.

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

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).

Pathogen	Antibody titres
Hepatitis B	37 ± 24 IU/g Ig
Hepatitis A	21 ± 4 IU/ml
Tetanus	16 ± 3 IU/ml
Cytomegalovirus	26 ± 5 UPEI/ml
Rubella	347 ± 54 IU/ml
Varicella	8 ± 0·9 IU/ml
Streptococcus pneumoniae	285 ± 22 mg/l
Haemophilus influenzae type b	15 ± 1 mg/l
Poliovirus type 1	0·39 ± 0·13 ratio sample/reference preparation
Measles	0·50 ± 0·07 ratio sample/reference preparation
Vaccinia	20·7 ± 7·2 IU/ml
Diphtheria	6 ± 1 U/ml

Ig: immunoglobulin.

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).

Parameter	Results
PKA (IU/ml)	< 2
ACA (CH50/mg Ig)	0·4 ± 0·1
Anti-A haemagglutinins	No agglutination Dil 1/4–1/32
Anti-B haemagglutinins	No agglutination Dil 1/4–1/16
Irregular antibodies	No agglutination Dil 1/2
Anti-D antibodies	Pass test
Osmolality (mOsm/kg)	329 ± 6
pH	5·6 ± 0·1
Total protein (mg/ml)	48 ± 1
Heat stability	Does not show any visible sign of gelation
Pyrogens	Pass test
Sterility	No microbiological growth
Abnormal toxicity	No indication of toxicity

ACA: anti-complementary activity; PKA: prekallikrein activator; Ig: immunoglobulin.

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’
Laboratory A			Laboratory B			Planova 20N
Run 1	Run 2	Run 3	Run 1	Run 2	Run 3	Run 1	Run 2	Run 3
1·30	1·13	0·65	1·04	0·65	1·00	4·45	3·86	4·79
Mean: 0·96						Mean: 4·37

Conclusions

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.

Conflicts of interest

J. I. Jorquera is an employee of Grifols S.A.