Accounting for the Donnan Effect in Diafiltration Optimization for High-Concentration UFDF Applications
The biopharmaceutical industry is targeting high-concentration protein formulations to enable subcutaneous administrations. Such administration can provide better patient convenience than intravenous administration. One challenge associated with high-concentration formulations is increased electrostatic interaction between proteins and excipients. That is a result of increased protein-charge density at high protein concentrations. Such interactions can create an offset between excipient levels in final products and diafiltration buffers in ultrafiltration processes. The effect of such electrostatic interactions in a membrane process is known as the Donnan effect.
Figure 1: ()
The Donnan effect on excipient levels has received significant attention in recent years. Theoretical modeling has been developed to predict excipient and pH changes as a result of the Donnan effect in monoclonal antibody (MAb) processes. One model based on the Poisson–Boltzmann equation provided good prediction of excipient levels in the final retentate pool (1). A second model developed by Bolton et al. demonstrated to be predictive for basic MAb and acidic Fc-fusion proteins (2). The latter study also included several mitigation strategies to achieve target levels of excipients at the end of an ultrafiltration–diafiltration (UFDF) process. Both publications provide tools for understanding the influence of the Donnan effect on target formulation excipients. By contrast, our study focuses on the influence of the Donnan effect on removal of starting buffer excipients during diafiltration.
PRODUCT FOCUS: HIGH-CONCENTRATION BIOLOGICS
PROCESS FOCUS: FORMULATION, FILL AND FINISH
WHO SHOULD READ: PROCESS DEVELOPMENT AND MANUFACTURING, FORMULATION, FILTRATION OPERATORS
KEYWORDS: EXCIPIENTS, AGGLOMERATION, BUFFER REMOVAL, DIAFILTRATION
LEVEL: INTERMEDIATE
A typical final-formulation UFDF step will target eight to 10 diavolumes. For an ideal process, in which excipients pass freely through the membrane (the retention value R = 0), 10 diavolumes provide 99.995% removal of starting excipients. That equates to a “complete” exchange (Figure 1).
Figure 1: ()
The Donnan effect, however, can influence that removal. We performed several test runs to demonstrate how the Donnan effect changes the removal efficiency of positively and negatively charged excipients. We conducted diafiltration test runs using a MAb at two different concentrations to additionally assess the influence of MAb concentration on excipient removal efficiency.
Materials and Methods
Protein: We used SAN-300, a MAb provided by Santarus Inc., for the diafiltration studies. It is a glycosylated IgG1 monoclonal antibody directed against VLA1 (very late antigen-1, α1β1 integrin). The protein is expressed by a Chinese hamster ovary (CHO) cell line and purified using a standard three-column MAb purification process. SAN-300 protein has pI >8 with a molecular weight >140 kDa.
Excipients: We studied three different excipients in subsequent experiments. Two were negatively charged organic-acid buffers (referred to here as EA– and EB–). The third excipient was positively charged (referred to here as E+).
UFDF Procedure: We perfomed all tests at room temperature (21 °C) using EMD Millipore Ultracel (regenerated cellulose) 30-kD membranes in 88-cm2 Pellicon 3 devices installed in a Pellicon Mini cassette holder. We ran diafiltrations at a transmembrane pressure (TMP) of 20 psig. The retentate was continuously stirred and recirculated through the system using a peristaltic pump. We performed initial concentration steps as needed to achieve the desired SAN-300 test concentration. Feed flow rate was 4.5 L/min/m2 for the runs. Table 1 provides a summary of the test matrix.
Table 1:
Table 1: 194; ()
Measurement of Protein Concentration: Spectrophotometric methods determined protein concentration using the extinction coefficient at 280 nm. We diluted the reference standard and sample to a specified concentration and then measured at A280, A320, and A360. We corrected the A280 reading for background absorbance at A320 and A360. We then calculated protein concentration using the corrected A280 dilution factor and extinction coefficient.
Excipient Assays: The positively charged excipient (E+) levels were determined using a capillary zone electrophoresis (CZE) method that uses a fused silica capillary (ID = 50 µm) with an enhanced detection cell, a borate electrolyte, and direct ultraviolet (UV) detection at 195 nm. We diluted the samples one hundredfold before analysis.
We measured the negatively charged excipients EA– and EB