Application of an aqueous two‐phase systems high‐throughput screening method to evaluate mAb HCP separation

Abstract

Aqueous two-phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud-point method for binodal determination was developed. PEG4000-PO(4) binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000-PO(4) systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task.

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

“…Aqueous two-phase system (ATPS) has shown to be considerably advantageous in terms of process economy and operation, especially when processing high titer cell culture supernatants [13]. This liquid-liquid extraction (LLE) technology has been successfully reported in recent times as a primary stage unit operation in the downstream processing of several biological products including therapeutic proteins, such as antibodies [4,10,11,[15][16][17][18][19][20][21][22][23][24]. Its technical feasibility at large scale has been, however, demonstrated for the downstream processing of just few biological products [25][26][27].…”

Optimization of PEG–salt aqueous two-phase systems by design of experiments

“…However, when the experimental region of interest is cuboidal rather than rotatable/ spherical or when it is preferable to maintain low and high factor levels, and still carry out a RSM design, the CCF or face-centered cube is a feasible alternative involving α = ± 1 [9,13], which is commonly used for the optimization of PEG-salt ATPS [2,16,17,22,25,28,31,34,36,[49][50][51][52][53][54][55][56][57]. For instance, in the CCF design for three factors (e.g., x 1 , x 2 , and x 3 ) the star/axial points are located on the centers of the faces of a cube or hypercube (Fig.…”

“…→ Table 1 The statistical significance of quadratic models or equations and their corresponding coefficients is generally evaluated by the analysis of variance (ANOVA) and standard least squares techniques [9,15,22,37], at which a multiple linear regression (MLR) analysis is usually performed in order to identify the coefficients of the significant effects of each response variable [22,53]. In this context, ANOVA is carried out for the determination of the significance and magnitude of the effects of variables and all their possible linear and quadratic interactions on a studied response variable by using Fisher's distribution or Fisher's statistical test (F-test), as discussed separately ahead [22].…”

“…Subsequently, the numerical sizes of variances or their estimations and the statistical significance of the obtained models and excluded parameters can be compared to each other and evaluated by performing the F-test for a set of parameters or output data set considering either F-or p-values of the model and the lack-of-fit (LOF) [13,22,53]. In this context, another common term in ANOVA is the mean squares (MS), which can be determined by the ratio of SS to the corresponding degrees of freedom (DF) [43].…”

Utilização de sistemas poliméricos de duas fases aquosas (SPDFA) compostos por polietileno glicol/ácido poliacrílico (PEG/APA) para extração de ácido clavulânico

“…Concerning VP1 and VP2 proteins, 95.3% or 33.2%, respectively, were recovered in the interface of a PEG 1000-magnesium sulfate system (Luechau et al, 2011). Ebrahimpour et al, (2010), used a chromatographic process of stable expanded beds which enables (Anspach et al, 1999) (Oelmeier et al, 2011).…”