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.

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

Development of a platform process for adenovirus purification that removes human SET and nucleolin and provides high purity vector for gene delivery.

The manufacturing of virus occurs at a modest scale in comparison to many therapeutic proteins mainly because a gene therapy dose is typically only µg of vector. Although modest in scale the generation of high purity virus is challenging due to low viral expression levels and the difficulties in adequately characterizing such a large and complex molecule. A 100 L bioreactor might produce only 100 mg of virus that must be separated from host and process impurities that are typically greater by several orders of magnitude. Furthermore, in the later purification stages the main milieu component is often virus at low concentration (µg/mL) which may non-specifically adsorb to purification surfaces resulting in a lowered virus recovery. This study describes our approach to develop a scalable, manufacturable robust process for an Adenovirus (Ad) gene therapy vector. A number of analytical tools were developed to guide the purification design. During process development, two human proteins, SET and nucleolin, were identified in viral preparations. To our knowledge, this is the first time that SET and nucleolin have been described in Ad. In this report we detail a process for their removal and the robust removal of all process, product and host cell impurities.

Periodic counter-current chromatography -- design and operational considerations for integrated and continuous purification of proteins.

Integrated and continuous processing of recombinant proteins offers several advantages over batch or semi-batch processing used traditionally in the biotechnology industry. This paper presents a theoretical and practical approach for designing a periodic counter-current chromatography (PCC) operation as a continuous capture purification step that is integrated with a perfusion cell culture process. The constraints for continuous and optimal PCC operation govern the selection of residence time and number of columns. The flexibility available in PCC design for selection of these parameters is dictated by the binding characteristics of the target protein on the capture resin. Using an empirical model for the protein breakthrough curve, analytical solutions to determine these conditions were derived and verified with experimental results for three different proteins: two relatively unstable proteins (recombinant enzymes) and a relatively stable protein (monoclonal antibody). The advantages of a continuous downstream capture step are highlighted for the three case studies in comparison with the existing batch chromatography processes. The use of PCC leads to improvements in process economics due to higher resin capacity utilization and correspondingly lower buffer consumption. Furthermore, integrated and continuous bioprocessing results in a smaller facility footprint by elimination of harvest hold vessels and clarification, as well as by reducing the capture column size by one to two orders of magnitude.

Integrated continuous production of recombinant therapeutic proteins.

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.

A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species.

Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.

Remodeling the oligosaccharides on ß-glucocerebrosidase using hydrophobic interaction chromatography and applications of hydroxyl ethyl starch for improving remodeling and enhancing protein stability.

In this article, we describe a hydrophobic interaction chromatography (HIC) method to remodel the carbohydrates on recombinant human ß-glucocerebrosidase (GCR) and the use of hydroxyl ethyl starch (HES) an ethylated starch polymer, to improve this process. GCR is a therapeutic protein used in the treatment of Gaucher disease, a life threatening condition in which patients lack sufficient functional levels of this enzyme. Gaucher disease is the most common inherited lysosomal storage disorder resulting in hepatomegaly, splenomegaly, and bone and lung pathology due to the accumulation of glucosylceramide in the lysosomes of macrophages (Beutler and Grabowski, 2001). The oligosaccharide remodeling of GCR, performed on HIC using three enzymes that remove sugars, increases macrophage uptake through the mannose receptor and thereby lowers its therapeutic dose versus unmodified GCR (Furbish et al., 1981; Van Patten et al., 2007). In this article we describe findings that the addition of HES lowered the amounts of three deglycosylating enzymes needed for remodeling GCR. HES also stabilized the activity of α-glucosidase, α-galactosidase, and GCR under conditions in which these three enzymes rapidly lose activity in the absence of this polymer. Circular dichroism (CD) and second derivative UV spectroscopy revealed that the secondary and tertiary structure of α-glucosidase was unchanged while for GCR there was a slight compaction of the secondary structure but no apparent affect on the tertiary structure. The thermal stability of both GCR and α-glucosidase were enhanced by HES as both molecules showed an increased transition midpoint (T(m)).

Lysozyme retention on hydrophobic interaction chromatography predicts resin performance at large scale.

It is critical that manufacturers understand the impact of resin variability on process performance and consistency. Choosing an appropriate resin lot often requires running manufacturing load material on a scale-down processing step and showing that the product recovery and purity is within manufacturing experience. In the present study, the LR (lysozyme retention value), on the vendor Certificate of Analysis, was predictive of the performance of an HIC (hydrophobic interaction chromatography) resin in a complex manufacturing step. This processing step is used to reduce host cell PIs (protein impurities) while maintaining a desired glycoform profile of recombinant TSH (thyroid-stimulating hormone). A correlation was found between a first-moment analysis of the HIC elution peaks and resin LR. Approximately 91% of the observed variation was accounted for by resin LR, and LRs of 55 and 50 were significantly different. The acceptable LR range, to maximize glycoform profile reproducibility and minimize recovery fluctuations, was dependent on the product collection method. Only resins with a narrow LR range within 1-2 units would be acceptable in a procedure in which set volume fractions are collected. Resins with an LR from 49 to 55 were appropriate when TSH collection was based on A280 ranges and this approach had the added benefit of collecting a single product pool.

The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery.

Flocculants have been employed for many years as aides in the clarification of wastewater, chemicals and food. Flocculants aggregate and agglutinate fine particles resulting in their settling from the liquid phase and a reduction in solution turbidity. These materials have not been widely used in the clarification of mammalian cell culture harvest. In this paper we examined chitosan as a flocculent of cells and cell particulates in NS0 culture harvest and the subsequent further clarification of this material by continuous flow centrifugation followed by depth and absolute filtration. Chitosan is an ideal flocculant for biotechnology applications as it is produced from non-mammalian sources (typically arthropod shells) and is also available in a highly purified form that is low in heavy metals, volatile organics and microbial materials. Chitosan is a polymer of deacetylated chitin. The deacetylation imparts limited solubility on insoluble chitin and the amino groups on the polymer result in a polycationic material at acidic and neutral pH that can interact with polyanions, such as DNA and cell culture debris (typically negatively charged). Likely the interaction of chitosan with cell culture particulate forms a germinal center for further interaction and agglomeration of particulates thereby reducing the solubility of these materials resulting in their settling out into the solid phase. Chitosan improved the clarification throughput six to seven folds without a deleterious effect on monoclonal antibody recovery or purity. The procedure for utilizing chitosan is facile, easily implemented, and highly effective in improving material clarity and increasing material throughput.