A pseudobiospecific hollow fiber cartridge for in vitro adsorption of autoantibodies from pathological serum

A PSEUDOBIOSPECIFIC HOLLOW FIBER CARTRIDGE FOR IN VITRO ADSORPTION OF AUTOANTIBODIES FROM PATHOLOGICAL SERUM

R.C.A.Ventura¹, R.L.Zollner², C.Legallais³,

M.A.Vijayalakshmi⁴ and S.M.A.Bueno1^*

1School of Chemical Engineering, State University of Campinas, Unicamp,

Campinas - SP, Brazil, E-mail:[e-mail]

²School of Medical Science, State University of Campinas, Unicamp,

Campinas - SP, Brazil, E-mail: [e-mail]

³URA CNRS 858, Laboratoire de Biomécanique et de Instrumentation Médicale,

Université de Technologie de Compiègne, B.P. 20649, F-60206 Compiègne,

France, E-mail: [e-mail]

⁴Laboratoire d'Interactions Moléculaires et de Technologie des Séparations,

Université de Technologie de Compiègne, B.P. 20649, F-60206

Compiègne Cedex, France, E-mail: [e-mail]

(Received: November 12, 1999 ; Accepted: May 18, 2000)

Abstract - The affinity filtration technique using histidine as a pseudobiospecific ligand immobilized on poly(ethylene vinyl alcohol) hollow fiber membranes (His-PEVA) was used to remove autoantibodies from serum of patients with autoimmune disease. The effects of buffering solution conditions on the efficiency of autoantibodies removal was studied. The removal of anti-dsDNA, anti-SS-A/Ro, anti-Sm, anti-Sm/RNP and anti-cardiolipin autoantibodies present in the serum was investigated, comparing the efficiency between Hepes and Tris-HCl buffers. The results showed the potential of the membrane to remove all the autoantibodies studied. Anti SS-A/Ro was removed more efficiently in Tris-HCl buffer system rather than with the Hepes buffer.

Keywords: autoantibodies, auto-immune diseases, extracorporeal circulation, histidine, affinity membranes

INTRODUCTION

Autoimmune diseases are characterized by the presence of abnormally high concentrations of autoantibodies (immunoglobulins mainly of the IgG class) and immune complexes. Corticosteroids and immunosuppressive agents have been a useful therapy for decades; however their application is limited by a number of well-known long-term side effects (Siegel, 1985). Plasma exchange has been applied to remove autoantibodies in the treatment of severe forms of various autoimmune diseases. Risks of plasma exchange therapy are related to the nonspecific elimination of plasma constituents other the disease-related autoantibody. Moreover, plasma exchange requires the use of plasma substitute, which is expensive and may potentially cause allergies, hypocalcemia, viral infections or other complications (Schneider et al., 1990).

Due to the disadvantages of plasma exchange, specific extracorporeal techniques to remove the pathogenic substances from plasma have been developed. Adsorbents with protein A as a ligand have been introduced in clinical apheresis to the selective removal of human IgG from plasma (Berta et al., 1994, Balint et al., 1995). The matrix-bound amino acids tryptophan and phenylalanine have been also applied as adsorbents in several immune disorders (Yamazaki et al., 1989; Schneider et al., 1990). In contrast to protein A, leakage of these ligands would not be followed by dangerous reactions (Yamazaki et al., 1989). However, the results of Ikonomov et al., 1992 showed a much lower adsorption for IgG in tryptophan and phenylalanine poly(vinyl alcohol) gels than that in protein A-Sepharose.

Histidine ligand affinity chromatography was employed for the purification of IgG from different sources (Vijayalakshmi, 1993). Histidine grafted membranes were demonstrated to be as excellent alternative to protein A coupled flat membrane. These membranes have capacities for human IgG equivalent to Protein A (Mandjiny and Vijayalakshmi, 1993).

L-histidine immobilized onto poly (ethylenevinyl alcohol) hollow fiber membranes (His-PEVA) showed high selectivity for IgG adsorption from human serum (Bueno et al., 1995, Bueno et al., 1996). In a recent work, it was observed that IgG anti-b₂ glycoprotein I and anti-prothrombin autoantibodies were retained on this functionalized membranes (Darnige et al., 1999). In addition, the physical and chemical stability of His-PEVA hollow fibers membranes allowed a good reusability and sterilization of the cartridge. The present work focuses on the in vitro feasibility of using histidine-linked hollow fiber membranes as a potential tool for the extracorporeal autoantibodies removal from patients with autoimmunes diseases.

MATERIALS AND METHODS

Materials

Sodium borohydride, L-histidine, 1,4 butanediol diglycidyl ether, hydroxyethylpiperazine ethanesulfonic acid (Hepes) were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. Ultrapure water was obtained using the Millipore Milli-Q (Millipore, Bedford, MA, USA). Poly(ethylene vinyl alcohol), PEVA, hollow fiber cartridges (Model Eval 4A, 1m² surface area) for blood plasma filtration were purchased from Kuraray (Osaka, Japan). The hollow fiber had an internal diameter of 200 mm, a wall thickness of 20 mm and a nominal molecular weight cut off of 600 kDa. Fibers were cut and assembled in a minicartridge of 4.5 cm effective length to yield a total of 0.1 g dry mass, 50.3 cm² surface area and 0.11 cm³ fiber bed volume.

Methods

Immobilization of L-Histidine onto PEVA Hollow Fiber Membranes

The PEVA hollow fiber membranes were activated with 1,4 butanediol diglycidyl ether and L-histidine was coupled as described by Bueno et al., 1995.

Tested Fluids

12 mL of serum was necessary for the affinity filtration experiments. Fresh frozen serum samples collected from patients with autoimmune disease were provided by Serum Bank of Laboratório de Imunologia Clínica (LICA, FCM, UNICAMP, Brazil). The samples were pooled before the experiments. Table 1 summarizes the content of pathological sera pools.

Serologic Analysis

Samples were collected from the tank after affinity filtration experiments and concentrations of IgG, IgM and IgA were determined nephelometrically (Array Protein System, Beckman Instruments, USA). Detection of antibodies to dsDNA, SS-A/Ro, Sm, Sm/RNP, RNP and cardiolipin was performed by an enzyme-linked immunosorbent assay (ELISA) system kit (Sanofi Diagnostics Pasteur, USA) according to the manufacturer’s instructions.

Affinity Filtration Experiments

In vitro experiments were carried out at room temperature (24-26°C). Prior to the experiments, 25 mM Hepes buffer, pH 7.0 or 25 mM Tris-HCl buffer, pH 7.4 were circulated during 15-20 min through the mini-cartridge. After that, the experiments were carried out by cross-flow filtration with recirculation of the retentate and the filtrate (closed-loop). The serum solution containing autoantibodies was pumped from the stirred tank by a peristaltic pump (Digi-staltic Masterflex) to the mini-cartridge and fed the filtration module. In order to keep the ratio Q_F/Q_i (filtrate flow rate/inlet flow rate) constant, the filtrate flow rate was adjusted through a second peristaltic pump (Bio-Rad Labs., Richmond, USA), (0.6 mL/min, corresponding to a residence time (t_R) of 11 sec). The residence time was calculated by dividing the membrane interstitial volume (0.11 cm³) by filtrate flow rate (0.6 mL/min). The filtrate outlet was monitored by UV detector at 280 nm (BioRad Labs., Richmond, USA) during the adsorption steps. Both filtrate and retentate lines were returned to the tank. The duration of each adsorption experiment was 1 h. The IgG, IgM, IgA or autoantibodies retaining capacity was defined as the mass which disappeared from the tank, normalized by the mini-cartridge surface area. The extensive washes to remove the unbound protein, were carried out following four steps : a dead-end filtration wash, a lumen wash, a shell wash and a backfushing wash (the filtrate compartment was filled with the equilibrium buffer and fractions were collected on the retentate line after crossing the fibers) as described by Bueno et al 1996. The effluents were monitored as described previously. The protein content in the tank’s solution were determined by ELISA and rate nephelometry. The His-PEVA hollow fiber mini-cartridge was regenerated by passing 1.5 M sodium hydroxide solution through the cartridge in cross-flow, frontal and backflushing modes.

RESULTS AND DISCUSSION

In our previous works, L-histidine immobilized onto poly (ethylene vinyl alcohol) hollow fiber membranes showed high selectivity for IgG adsorption from human serum. The amount of albumin adsorbed in His-PEVA was very small (Bueno et al., 1995). IgG adsorption onto His-PEVA affinity membranes depends on the buffer system used. Adsorption was possible over a broad pH range and was found to depend strongly on the nature and protonation state of the buffer ions. For example, Mes, Mops and Hepes buffers allowed the highest adsorption of IgG despite the fact that the ionic strengths of these solutions were higher than those of acetate, Tris.HCl and phosphate. For instance, in Hepes buffer at pH 7.0, IgG₁, IgG₂ and IgG₃ were bound, whereas in Tris-HCl buffer pH 7.4, only IgG₁ and a part of the IgG₃ fraction was retained (Haupt et al., 1995).

Autoantibodies retention studies were carried out using Tris-HCl and Hepes buffers at pH 7.4 and 7.0, respectively. Hepes (zwitterionic) and Tris-HCl (non-zwiterionic) buffer were chosen as representatives of two groups of buffers (Haupt et al., 1995). Table 2 shows autoantibodies retention capacity on His-PEVA in both buffers used. Figure 1 shows the graphic presentation of these results for pool 5.

Retention of anti-dsDNA, anti-SS-A/Ro, anti-Sm, anti-Sm/RNP and anti-cardiolipin antibodies in His-PEVA affinity membrane was possible in both buffers studied. Comparing the results of the experiments carried out at differents pools of autoantibodies, no significant difference was observed in the relative retention of anti-dsDNA, anti-Sm, anti-Sm/RNP and anti-cardiolipin antibodies when Tris-HCl or Hepes buffers were used. However, Tris-HCl buffer allowed a removal capacity from 50 to 117 % higher than the Hepes buffer.

The removed proteins were analyzed by rate nephelometry to quantify IgA, IgG, IgM (Table 3). The low amount of IgG removed using Tris-HCl buffer is due to the fact that the whole IgG fraction is not removed from serum (only IgG₁ and IgG₃ are adsorbed) (Haupt et al., 1995). Compared to IgG, IgM showed a significant retention percentage, while the amount of IgA retained in all tests was significantly smaller than IgG.

CONCLUSION

This work shows that the amino acid histidine coupled to PEVA hollow fiber membrane constitutes a potential tool for IgG and IgM removal from human plasma if buffer exchange and ultrafiltration steps are included in the extracorporeal circuit.

The approach proposed may offer a useful alternative to protein A based devices in treatment of immune-related diseases. Moreover, as the system can be fine-tuned (by using the appropriate buffer) to improve the adsorption of specific autoantibodies and the pathogenic immunoglobulins from the patient’s blood.

In the process of selective adsorption aiming autoantibodies removal, is interesting to study which proteins are the most adsorbed in percentage terms. Among the immunoglobulins studied (IgA, IgG, IgM) our results demonstrated a considerable retention of IgG, IgM, in contrast to IgA, by His-PEVA. However, the exact mechanism of adsorption remain poorly understood.

The high capacity, specificity and stability for immunoglobulins removal, incluse autoantibodies, demonstrated by His-PEVA, associated to their lower cost, make a useful alternative system to remove undesireble serum pathogenic proteins. However, further experiments under real conditions are necessary in order to evaluate this system in treatment of patients.

ACKNOWLEDGEMENTS

We are very grateful to the PADCT, FAPESP and FAEP, Brazil, for financial support.

NOMECLATURE

C_i initial autoantibodies concentration in the reservoir (IU/mL) C autoantibodies concentration in the reservoir after 1 h (IU/mL) Le effective length of the hollow fibers (cm) N_f number of hollow fibers in the cartridge Q_F filtrate flow rate (mL/min) Q_i inlet flow rate (mL/min) Q_R autoantibodies removing capacity (IU/cm²) R autoantibodies retained/autoantibodies loaded x 100

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*

To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  16 Mar 2001
• Date of issue
  Dec 2000

History

• Accepted
  18 May 2000
• Received
  12 Nov 1999