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.
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.
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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).
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 . The presence of fractions indicates degradation of the molecule . 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 .
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 .
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).
|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|
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).
|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|
|Sterility||No microbiological growth|
|Abnormal toxicity||No 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 A||Laboratory B|
|Run 1||Run 2||Run 3||Run 1||Run 2||Run 3||Run 1||Run 2||Run 3|
|Mean: 0·96|| || || ||Mean: 4·37|