Determination of the residual hemolytic activity of a purified bacteriophage suspension

M. Pichon-Pramil1, B. Lapras1, M. Medina2, M. Cristofoli1, C. Marchand1, C. Merienne1, F. Laurent2, F. Pirot1
1 Unité de Préparation et de Contrôle des Médicaments, plateforme FRIPHARM, Pharmacie à Usage Intérieur, Groupement Hospitalier Edouard Herriot – Hospices Civils de Lyon
2 Institut des Agents Infectieux, Laboratoire de Biologie et d’Anatomie Pathologique, Groupement Hospitalier Croix Rousse - Hospices Civils de Lyon

Phage therapy is the use of bacteriophages (BP) for clinical uses. It gains interest as an option against antibioresistance. The production steps involves the amplification of phages within the bacteria, which are lysed, releasing BP and toxins in the medium. For anti-Staphylococcus aureus BP, the purification step needs to remove efficiently hemolysin from the BP suspension. In this work, we report the development of a UV-Visible spectrophotometric detection method of hemolysis.

ASTM considers that a product is hemolytic if its hemolytic index HI(%) is greater than 5.0% [2]. HI is calculated as follows: HI=100[(CS-CB)/CC]. CS: Hemoglobin (Hb) concentration of a red blood cells suspension exposed to BP suspension (test solution S); CC: Hb concentration of a completely hemolysed red blood cells suspension (positive control C); CB: Hb concentration of blank B.

To calculate CS: a suspension of red blood cells was exposed to a suspension of BP. The resulting suspension were incubated 3 h at 37°C. If the BP suspension contains hemolysin then red blood cells are lysed, releasing hemoglobin (Hb) in the medium. After incubation, the suspension were centrifuged to separate red blood cells (bottom) and hemoglobin (supernatant). The supernatant reacted with Drabkin® solution (D). D transforms hemoglobin into cyanmethemoglobin, which absorbs light at 540 nm. It allowed us to determine the concentration of Hb released. A blank B was realised using the same method as S. However, instead of using a BP suspension, PBS was used. To ensure the accuracy of the method, ranges between 0% and 10% of controlled hemolysis were achieved by dilutions of Brij® 0.5% (Brij® 30% diluted in purified water) in NaCl 0.9%. In this work, the red blood cells come from PBS-washed sheep blood.

A range of 5 points of controlled hemolysis was achieved by induced lysis of red blood cells suspensions mixed with 0.0, 3.0, 5.0, 7.0, 10.0 and 100.0% (v/v) of Brij® 0.5%. The mean and 95% confidence interval of the HI – induced by Brij® 0.5% from 3.0% to 10.0% – are respectively of 2.3% [0.6; 4.0], 6.0% [2.5; 9.5], 7.2% [2.1; 12.3], 12.2% [0.4; 24.1]. The linear correlation coefficient R² - for solutions of Brij® 0.5% from 0.0 to 10.0% - was 0.949. There is therefore a linear correlation between the Brij® 0.5% concentration and the induced hemolysis.
B, C and S solutions will be implemented in a second phase.

Discussion and conclusion
Despite low accuracy (CV=85.6%), the method shows linearity between HI and Brij® 0.5% concentration. Method validation will be performed with diluted hemolysin exposed for 3h at 37°C to human’s red blood cells suspension. This could significantly improve the accuracy of controlled hemolysis ranges. After validation, this method will be included in the QC of anti-S. aureus BP suspensions after purification.

[1] : Ferry T, Kolenda C, Batailler C and al. Phage Therapy as Adjuvant to Conservative Surgery and Antibiotics to Salvage Patients With Relapsing S. aureus Prosthetic Knee Infection. Front Med. 2020 nov 16;7:570572.
[2] : ASTM. Standard Practice for Assessment of Hemolytic Properties of Materials. Book of Standards. 2017 ; 13.01 :6

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