ce with U.S. regulatory requirements. As an additional safety measure, mini-pools of the plasma are tested for the presence of HIV-1 and HCV by FDA licensed Nucleic Acid Testing (NAT).
To further improve the margin of safety, three dedicated, independent and effective virus inactivation/removal steps have been integrated into the manufacturing and formulation processes, namely solvent/detergent (S/D) treatment, 35 nm nanofiltration,and a low pH incubation at elevated temperature. The S/D process includes treatment with an organic mixture of tri‑n-butyl phosphate, octoxynol 9 and polysorbate 80 at 18°C to 25°C for a minimum of 60 minutes7.
In vitro virus spiking studies have been used to validate the capability of the manufacturing process to inactivate and remove viruses. To establish the minimum applicable virus clearance capacity of the manufacturing process, these virus clearance studies were performed under extreme conditions (e.g., at minimum S/D concentrations, incubation time and temperature for the S/D treatment). Virus clearance studies for the Immune Globulin Infusion 10% (Human) of HYQVIA performed in accordance with good laboratory practices (Table 6) have demonstrated that:
•
S/D treatment inactivates the lipid-enveloped viruses investigated to below detection limits within minutes.
•
35 nm nanofiltration removes lipid-enveloped viruses to below detection limits and reduces the non‑lipid enveloped viruses HAV and B19V. As determined by a polymerase chain reaction assay, nanofiltration reduced B19V by a mean log10 reduction factor of 4.8 genome equivalents.
•
Treatment with low pH at elevated temperature of 30°C to 32°C inactivates lipid-enveloped viruses and encephalomyocarditis virus (EMCV, model for HAV) to below detection limits, and reduces mice minute virus (MMV, model for B19V).
Table 6 Three Dedicated Independent Virus Inactivation/Removal Steps Mean Log10 Reduction Factorsa (RFs) For Each Virus and Manufacturing Step Virus type Enveloped
RNA Enveloped DNA Non-enveloped
RNA Non-enveloped
DNA
Family Retroviridae Flaviviridae Herpesviridae Picornaviridae Parvoviridae
Virus HIV-1 BVDV WNV PRV HAV EMCV MMV
SD treatment
> 4.5
> 6.2
n.a.
> 4.8
n.d.
n.d.
n.d
35 nm nanofiltration
> 4.5
> 5.1
> 6.2
> 5.6
5.7
1.4
2.0
Low pH treatment
> 5.8
> 5.5
> 6.0
> 6.5
n.d.b
> 6.3
3.1
Overall log reduction factor (ORF)
> 14.8
> 16.8
> 12.2
> 16.9
5.7 b
> 7.7
5.1
Abbreviations: HIV-1, Human Immunodeficiency Virus Type 1; BVDV, Bovine Viral Diarrhea Virus (model for Hepatitis C Virus and other lipid enveloped RNA viruses); WNV, West Nile Virus; PRV, Pseudorabies Virus (model for lipid enveloped DNA viruses, including Hepatitis B Virus); EMCV, Encephalomyocarditis Virus (model for non-lipid enveloped RNA viruses, including Hepatitis A virus [HAV]); MMV, Mice Minute Virus (model for non-lipid enveloped DNA viruses, including B19 virus [B19V]); n.d. (not done), n.a. (not applicable).
a For the calculation of these RF data from virus clearance study
