Conjugated Nonionic Detergent Micelles: An Efficient Purification Platform for Dimeric Human Immunoglobulin A

Abstract

The SARS-COV-2 virus is a deadly agent of inflammatory respiratory disease. Since 2020, studies have focused on developing new therapies based on galactose-rich IgA antibodies. Clinical surveys have also revealed that galactose-deficient IgA1 polymerizes in serum, producing IgA nephropathy, which is a common cause of kidney failure in young adults. Here we show that IgA1-IgA2 dimers are efficiently and economically purified in solution via conjugated nonionic surfactant micellar aggregates. Quantitative capture at pH 7 and extraction at pH 6.5 can avoid antibody exposure to acidic, potentially denaturing conditions. Brij-O20 aggregates lead to the highest process yields (88-91%) and purity (94%). Recovered IgA dimers preserve their native secondary structure and do not self-associate. Increasing the reaction volume has little impact on yield or purity. By introducing an efficient, inexpensive IgA purification protocol, we assist pharmaceutical firms and research laboratories in developing new IgA-based therapies as well as in increasing our understanding of IgA1 polymerization.

Figure 1

A. Reagents used for the preparation of conjugated surfactant micelle aggregates. Surfactant #1 is the nonionic surfactant Tween-20 (Polysorbate 20), Brij-O20, or Triton X-100. Surfactant #2 is DDM, a low molecular weight surfactant with a hydrophilic maltose headgroup. For simplicity, the surfactant mixed micelles are drawn as spherical, although they may actually be ellipsoidal. Micelles are specifically conjugated upon addition of the [(bathophenanthroline)₃:Fe²⁺] complex, leading to surfactant micellar aggregates. Addition of tyrosine monomers, which preferentially interact with the hydrophobic micellar core, has been shown to allow extraction of antibodies at close to neutral pH. B. Scheme of a two-step human IgA dimer purification protocol based on conjugated micellar aggregates. Prior to capture, IgA is mixed with an artificial impurity dispersion containing bacterial proteins, i.e., E. coli lysate. Scheme drawings are not to scale.

Figure 2

SDS-PAGE of dIgA recovered from Brij-O20/DDM surfactant aggregates under different conditions: A. Effect of buffer (NaPi) concentration on dIgA recovery at pH 6.5 following 30 min incubation at 10 °C. B–D. Effects of temperature, pH, and time on IgA recovery in the presence of 200 mM NaPi. When the temperature, pH, or time was independently varied, the remaining parameters were set as indicated in A. IgA yields are indicated below the gel lane numbers and were quantitated using ImageJ (NIH). Gels are Coomassie stained.

Figure 3

SDS-PAGE demonstrates reproducibility of purification of IgA dimers using conjugated Tween-20 (A) or Triton X-100 (B) nonionic surfactant mixed micelles (with DDM, Tyr monomers). dIgA was captured in the absence of E. coli lysate, as described in the Supporting Information, and extracted within 30 min at 10 °C in the presence of 100 mM NaPi (pH 6.5). Total yield (%) is listed below the lane number. The control [C] in lane 2 was commercial dIgA. SDS-PAGE gels are Coomassie stained. Quantitation was accomplished using ImageJ (NIH).

Figure 4

Native-PAGE. dIgA was captured in the absence of E. coli lysate, as described in the Supporting Information, and extracted after 30 min in the presence of 100 mM NaPi (pH 6.5) at 10 °C. Recovered IgA was then run on the native PAGE gel. Lane 1: molecular weight markers; lanes 2–4: dIgA purified with surfactant aggregates; lane 5: [C] control. The gel is Coomassie stained.

Figure 5

Hydrodynamic particle size distribution of dimeric IgA purified in the absence of E. coli lysate with the conjugated surfactant mixed micellar platform as determined by DLS (see the Supporting Information for details). A: Brij-O20; B: Triton X-100; and C: Tween-20. In each panel, the control, i.e., commercial IgA, is denoted by a dashed black curve. Conditions: dIgA, ∼0.3 mg/mL; buffer, 100 mM NaPi, pH 6.5; measurement temperature, 25 °C.

Figure 6

Far-UV circular dichroism (CD) spectra of dimeric IgA, purified in the absence of E. coli lysate, using the conjugated nonionic surfactant mixed micelle platform prepared as described in the Supporting Information. A: Brij-O20; B: Triton X-100; and C: Tween-20. Measurements were performed at 25 °C; antibody concentration was 0.1 mg/mL in 100 mM NaPi buffer (pH 6.5). Commercial dIgA was measured as the control and is denoted by the blue curve in each panel. In panel B, the control spectrum was translated vertically by a constant amount for ease of viewing.

Figure 7

Comparison of efficiency of dIgA recovery (yield % dIgA) in the presence of E. coli lysate as determined by SDS-PAGE for each of the three nonionic surfactant mixed micellar platforms (+DDM; +Tyr) under study. Lane 1: molecular weight markers; Lanes 2–3: controls - commercial dIgA without (lane 2) or with (lane 3) E. coli lysate; Lanes 4–5, 6–7, and 8–9: dIgA, recovered from the conjugated surfactant mixed micellar aggregates following 30 min incubation at 10 °C in the presence of 100 mM NaPi (pH 6.5). This concentration was determined to be optimal in the presence of an E. coli lysate. Recovery yields are indicated below lane numbers and were quantitated using ImageJ (NIH). The gels are Coomassie blue stained.