Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display

Abstract

Background: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display.

Material and methods: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count.

Results: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity.

Discussion: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

Figure 1. Blood filter aggregate analysis

(A) Photograph of the disposable donor harness installed in the Haemonetics PCS2 Plasma collection system after a plasma donation. (B) Diagram representing where the blood filter chamber is positioned for the blood draw (black arrow) and red cell return (red arrow) during the plasma donation (not to scale). (C) Photograph of the two different blood filter chamber presentations observed after the plasma donation of 10 anti-D donors. From each blood filter chamber of 10 anti-D donors, aggregates were absent in 5 blood filters (C1) and aggregates were present in 5 blood filter chambers (C2) (D) Wright-Giemsa smears of blood filter aggregates for donor 1 and donor 2 (×40 objective). Lymphocytes (green arrows) were identified in the blood filter aggregates despite differing amounts of platelet aggregates (black arrows) and RBCs (red arrows). Fibrin (blue arrows) were also observed. (E) Blood count summary from a haemotological analyser and manual differential count. Significant differences in RBCs and platelet counts were observed between the 2 anti-D donors.

Figure 2. Reactivity profile of reformatted monoclonal antibodies against screening cells

Flow histograms showing the reactivity of an anti-D monoclonal antibody clone R593, a secondary only control and 4 reformatted mAbs (clone 2 to 5) against a commercial screening cell panel comprising of red cells with three different phenotypes: R1R1 (blue), R2R2 (red) and rr (black).