Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

Abstract

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody-polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to < 1 microg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody.

Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition

“…So far, these processes have usually been performed with precipitating agents such as PEG, polyvinylsulfonic acid (PVS), polyacrylic acid (PAA) and polystyrenesulfonic acid (PSS). These macromolecules are required to be subsequently removed (45,46). However, in the case of antibodies having LLPS properties as MAb A does, the purification and concentration steps can be completed without using precipitating agents.…”

Aggregative protein–polyelectrolyte complex for high-concentration formulation of protein drugs

“…The surface charge of a protein is mainly determined by the isoelectric point (pI) and pH: when pH < pI, the protein has a positive charge and when pH > pI, the protein has a negative charged. Note that aggregative PPC of antibody was found to be empirically favorable in the pH range of | pH − pI | ≈ 2 [18,36]. In addition, the aggregative PPC prepared by this condition more salt-soluble than that of other conditions (i.e.…”

“…The salt responsibility of PPC is generally independent of the state of the PPC. The release of protein molecules from a PPC is influenced not only by the incremental increase in ionic strength but also by the addition of other electrolyte, including polyelectrolytes and proteins, and hence are used in several applications, such as purification [36,[41][42][43][44][45], enzyme switch [24,[30][31][32], biosensors [46][47][48], and drug delivery systems [25,49,50]. 6 …”

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

“…Precipitation using polyelectrolytes can be implemented to selectively recover target proteins [7,8] or to remove impurities while leaving the protein product in the solution [9]. Polyelectrolytes are effective in precipitating proteins because of the interaction between the charges on the proteins and on the polyelectrolytes, creating insoluble complexes, and subsequent bridging of insoluble complexes either by residual charge interaction or through hydrophobic patches on the complexes to form larger clusters [5].…”