Advanced Analysis of Biosensor Data for SARS-CoV-2 RBD and ACE2 Interactions.

The traditional approach for analyzing interaction data from biosensors instruments is based on the simplified assumption that also larger biomolecules interactions are homogeneous. It was recently reported that the human receptor angiotensin-converting enzyme 2 (ACE2) plays a key role for capturing SARS-CoV-2 into the human target body, and binding studies were performed using biosensors techniques based on surface plasmon resonance and bio-layer interferometry. The published affinity constants for the interactions, derived using the traditional approach, described a single interaction between ACE2 and the SARS-CoV-2 receptor binding domain (RBD). We reanalyzed these data sets using our advanced four-step approach based on an adaptive interaction distribution algorithm (AIDA) that accounts for the great complexity of larger biomolecules and gives a two-dimensional distribution of association and dissociation rate constants. Our results showed that in both cases the standard assumption about a single interaction was erroneous, and in one of the cases, the value of the affinity constant KD differed more than 300% between the reported value and our calculation. This information can prove very useful in providing mechanistic information and insights about the mechanism of interactions between ACE2 and SARS-CoV-2 RBD or similar systems.

Affinity Chromatography: An Enabling Technology for Large-Scale Bioprocessing.

Affinity chromatography (AC) has been used in large-scale bioprocessing for almost 40 years and is considered the preferred method for primary capture in downstream processing of various types of biopharmaceuticals. The objective of this mini-review is to provide an overview of a) the history of bioprocess AC, b) the current state of platform processes based on affinity capture steps, c) the maturing field of custom developed bioprocess affinity resins, d) the advantages of affinity capture-based downstream processing in comparison to other forms of chromatography, and e) the future direction for bioprocess scale AC. The use of AC can result in economic advantages by enabling the standardization of process development and the manufacturing processes and the use of continuous operations in flexible multiproduct production suites. These concepts are discussed from a growing field of custom affinity bioprocess resin perspective. The custom affinity resins not only address the need for a capture resin for non-platformable processes, but also can be employed in polishing applications, where they are used to define and control drug substance composition by separating specific product variants from the desired product form.

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities.

Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer's Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations.

High-throughput process development of chromatography steps: advantages and limitations of different formats used.

In the past, development of a chromatographic separation method has been accomplished by performing a series of experiments using either manual or automated chromatography systems. The screening of a vast experimental space became very expensive because all experiments had to be performed in a serial manner, and the chromatography systems used were designed for relatively large columns and, therefore, the experiments required large sample volumes. To address these issues, high-throughput miniaturized methods employing different operating principles and/or formats have been introduced. Herein, a technical review of the most common high-throughput formats used for the development of chromatographic purification steps is presented. The formats considered include minicolumns, prefilled pipette tips, and microtiter filter plates prefilled with chromatography resins. Advantages and limitations of each format are discussed through the prism of chromatographic theory, engineering principles, and known mass-transfer mechanisms. A roadmap for applicability of the different formats for process development purposes and implementation of a Quality by Design initiative for designing/optimization of chromatography steps is also discussed.

High-throughput process development: determination of dynamic binding capacity using microtiter filter plates filled with chromatography resin.

Steadily increasing demand for more efficient and more affordable biomolecule-based therapies put a significant burden on biopharma companies to reduce the cost of R&D activities associated with introduction of a new drug to the market. Reducing the time required to develop a purification process would be one option to address the high cost issue. The reduction in time can be accomplished if more efficient methods/tools are available for process development work, including high-throughput techniques. This paper addresses the transitions from traditional column-based process development to a modern high-throughput approach utilizing microtiter filter plates filled with a well-defined volume of chromatography resin. The approach is based on implementing the well-known batch uptake principle into microtiter plate geometry. Two variants of the proposed approach, allowing for either qualitative or quantitative estimation of dynamic binding capacity as a function of residence time, are described. Examples of quantitative estimation of dynamic binding capacities of human polyclonal IgG on MabSelect SuRe and of qualitative estimation of dynamic binding capacity of amyloglucosidase on a prototype of Capto DEAE weak ion exchanger are given. The proposed high-throughput method for determination of dynamic binding capacity significantly reduces time and sample consumption as compared to a traditional method utilizing packed chromatography columns without sacrificing the accuracy of data obtained.

A methodology for the comparative evaluation of alternative bioseparation technologies.

Advances in upstream technologies and growing commercial demand have led to cell culture processes of ever larger volumes and expressing at higher product titers. This has increased the burden on downstream processing. Concerns regarding the capacity limitations of packed-bed chromatography have led process engineers to begin investigating new bioseparation techniques that may be considered as "alternatives" to chromatography, and which could potentially offer higher processing capacities but at a lower cost. With the wide range of alternatives, which are currently available, each with their own strengths and inherent limitations, coupled with the time pressures associated with process development, the challenge for process engineers is to determine which technologies are most worth investigating. This article presents a methodology based on a multiattribute decision making (MADM) analysis approach, utilizing both quantitative and qualitative data, which can be used to determine the "industrial attractiveness" of bioseparation technologies, accounting for trade-offs between their strengths and weaknesses. By including packed-bed chromatography in the analysis as a reference point, it was possible to determine the alternatives, which show the most promise for use in large-scale manufacturing processes. The results of this analysis show that although the majority of alternative techniques offer certain advantages over conventional packed-bed chromatography, their attractiveness overall means that currently none of these technologies may be considered as viable alternatives to chromatography. The methodology introduced in this study may be used to gain significant quantitative insight as to the key areas in which improvements are required for each technique, and thus may be used as a tool to aid in further technological development.

Ion exchange chromatography of antibody fragments.

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.