Precision analysis for the determination of steric mass action parameters using eight tobacco host cell proteins

The use of predictive models to develop chromatography-based purification processes

“…Often, modified versions of kinetic equations are used to reduce or increase the complexity of the mechanistic model e.g., as shown for the extended Langmuir model or exponentially modified Langmuir model ( Table 1 ). The SMA model is often used to describe sorption during ion exchange chromatography (IEX) and specifically accounts for the salt concentration, number of interacting ligands, and steric shielding of the ligand by bound proteins ( Parente and Wetlaufer 1986 ; Degerman et al, 2007 ; Osberghaus et al, 2012a ; Bernau et al, 2021 ). Currently, more than 15 isotherms are available for the description of chromatography modalities such as IEX and HIC ( Guo et al, 2020 ; Kumar and Lenhoff 2020 ; Saleh et al, 2021a ; Saleh et al, 2021b ; Kumar et al, 2021 ) as has been reviewed elsewhere ( Wang et al, 2016 ; Shekhawat and Rathore 2019 ).…”

“…For example, an inaccurately determined column porosity can distorted the value calculated for the equilibrium constant when fitting a chromatography model to a given peak ( Heymann et al, 2022 ). Various methods have been proposed to determine equilibrium parameters ( Shukla et al, 1998 ; Osberghaus et al, 2012a ; Bernau et al, 2021 ) but the experimental determination of different types of liquid volumes in columns remains challenging, especially for packed-bed chromatography using porous, spherical beads as the stationary phase. These liquid volumes consist of inter-particle and intra-particle components ( Figure 1 ) ( Frank et al, 2022 ).…”

“…The system void volume is usually determined by replacing the column with a zero-volume connector and then injecting an acetone pulse ( Schmidt-Traub et al, 2012 ; Marek et al, 2018 ; Desmet and Broeckhoven 2019 ; Bernau et al, 2021 ). The system void volume can then be calculated by multiplying the time difference between injection and detection at a UV monitor by the volumetric flow rate.…”

“…One widely-used approach is the inverse method ( Osberghaus et al, 2012a ; Hahn et al, 2016a ; Hahn et al, 2016b ), which fits isotherm parameters to an experimental elution peak by minimizing the discrepancies between experimental and simulated peaks ( Dose et al, 1991 ; Leweke and Lieres 2018 ). Typically, two chromatograms are necessary to fit, for example, the equilibrium constant k eq and the characteristic charge ν of the SMA isotherm ( Osberghaus et al, 2012a ; Bernau et al, 2021 ). The amount of protein required for such an experimental series is ∼0.6 mg, depending on the quality of the UV monitor.…”

“…Using competitive isotherms for multicomponent elution as well as fluorescent proteins or tags alleviates the need for highly pure protein during model calibration ( Seidel-Morgenstern 2004 ; Baumann et al, 2015 ). In our hands, purities of >50% are typically required to obtain a Gaussian curve-shaped peak for fitting ( Bernau et al, 2021 ), yet higher purities may be necessary depending on the type, number and individual abundance of non-target protein impurities. The presence of such impurities may result in shoulders or even several peak maxima, which in the simplest case can falsify the fitted transport parameters, and in the worst case can result in incorrect protein-specific isotherm parameters due to the selection of inappropriate peak properties, e.g., maxima, for fitting ( Pirrung et al, 2018 ).…”