Thomas O. Linden

A practical strategy for using miniature chromatography columns in a standardized high-throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance.

Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.

Targeted purification development enabled by computational biophysical modeling

Chromatographic and non‐chromatographic purification of biopharmaceuticals depend on the interactions between protein molecules and a solid–liquid interface. These interactions are dominated by the protein–surface properties, which are a function of protein sequence, structure, and dynamics. In addition, protein–surface properties are critical for in vivo recognition and activation, thus, purification strategies should strive to preserve structural integrity and retain desired pharmacological efficacy. Other factors such as surface diffusion, pore diffusion, and film mass transfer can impact chromatographic separation and resin design. The key factors that impact non‐chromatographic separations (e.g., solubility, ligand affinity, charges and hydrophobic clusters, and molecular dynamics) are readily amenable to computational modeling and can enhance the understanding of protein chromatographic. Previously published studies have used computational methods such as quantitative structure–activity relationship (QSAR) or quantitative structure–property relationship (QSPR) to identify and rank order affinity ligands based on their potential to effectively bind and separate a desired biopharmaceutical from host cell protein (HCP) and other impurities. The challenge in the application of such an approach is to discern key yet subtle differences in ligands and proteins that influence biologics purification. Using a relatively small molecular weight protein (insulin), this research overcame limitations of previous modeling efforts by utilizing atomic level detail for the modeling of protein–ligand interactions, effectively leveraging and extending previous research on drug target discovery. These principles were applied to the purification of different commercially available insulin variants. The ability of these computational models to correlate directionally with empirical observation is demonstrated for several insulin systems over a range of purification challenges including resolution of subtle product variants (amino acid misincorporations). Broader application of this methodology in bioprocess development may enhance and speed the development of a robust purification platform.

Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale.

High-throughput techniques to evaluate the effect of ligand density for impurity separations with multimodal cation exchange resins

Scale‐down, high‐throughput screening techniques are well on their way to becoming a commodity in downstream bioprocess development, especially for the rapid development of chromatography process steps. This work used both resin slurry plate and miniature column high‐throughput screening methodologies to identify the best resin properties for mAb separations utilizing a multimodal chromatography ligand interaction. A ligand with both cation exchange and hydrophobic interaction properties was studied at several ligand densities and compared to a commercially available multimodal resin with a larger particle size at high ligand density. The resins were screened with mAbs containing distinct process impurities (aggregates and a hydrophobic variant), and optimized conditions provided more than a log of clearance of both types of impurities for the different resins screened. These studies reveal that while a smaller particle size is generally preferable, optimal ligand densities can be different depending on the properties of both the mAb and impurity studied.

Domain antibody downstream process optimization: High-throughput strategy and analytical methods

Application of scale‐down high‐throughput screening has become integral for process development of antibody therapeutic products. In this work, methods are described for using high‐throughput techniques to develop a multicolumn chromatography purification protocol for a small domain antibody with very limited material (<200 mg). Screenings utilized resin slurry plates to explore and narrow potential operating space, and miniature columns were used to either confirm operating spaces or further explore impurity separations. Lab scale column confirmations were performed when appropriate. Affinity capture chromatography as well as ion exchange and multimodal polishing chromatography steps were explored. Feedstreams were pooled and recycled to preserve material for the different chromatography steps. Precise high‐throughput analytical assays were developed to fully characterize the domain antibody to a similar extent as a typical commercial therapeutic protein program. Optimized two‐column and three‐column processes provided overall chromatography yields of 66 and 58%, respectively, and were able to meet typical early phase requirements for removal of impurities such as aggregates, host cell protein, endotoxin, and other product‐related impurities. This study provides a comprehensive example of how a thorough biologics downstream process can be developed with a minimum of material.

High throughput chromatography strategies for potential use in the formal process characterization of a monoclonal antibody

High throughput experimental strategies are central to the rapid optimization of biologics purification processes. In this work, we extend common high throughput technologies towards the characterization of a multi‐column chromatography process for a monoclonal antibody (mAb). Scale‐down strategies were first evaluated by comparing breakthrough, retention, and performance (yields and clearance of aggregates and host cell protein) across miniature and lab scale columns. The process operating space was then evaluated using several integrated formats, with batch experimentation to define process testing ranges, miniature columns to evaluate the operating space, and comparison to traditional scale columns to establish scale‐up correlations and verify the determined operating space. When compared to an independent characterization study at traditional lab column scale, the high throughput approach identified the same control parameters and similar process sensitivity. Importantly, the high throughput approach significantly decreased time and material needs while improving prediction robustness. Miniature columns and manufacturing scale centerpoint data comparisons support the validity of this approach, making the high throughput strategy an attractive and appropriate scale‐down tool for the formal characterization of biotherapeutic processes in the future if regulatory acceptance of the miniature column data can be achieved. Biotechnol. Bioeng. 2016;113: 1273–1283.

High‐throughput purification tools for rapid upstream process development are interchangeable for biologics

Upstream process development of biologics is not only productivity‐driven but also quality‐driven. Typically, most quality attributes are not directly measurable in cell culture samples due to low product concentration and purity, thus requiring some level of sample purification. As higher throughput upstream technologies become available, sample purification is becoming a bottleneck in limiting the number and types of cell culture samples that can be analyzed. The application of high‐throughput, microscale protein purification techniques has the potential to address and expand this capability. In this work, the affinity capture step of an IgG1 mAb was adapted to fit resin‐plate, resin‐tip, and mini‐column formats in an attempt to approximate the packed‐column performance by optimizing parameters such as contact time, liquid/resin ratio, and loading to produce a yield of ≥70% yield. A representative cell culture supernatant was purified using both the optimized microscale and conventional techniques, and analyzed using a comprehensive panel of product quality assays. This direct comparison demonstrated that each technique generates product of equivalent purity across a wide range of feed conditions. The analytical comparability suggests that any of the conventional and high‐throughput methods are interchangeable for biologics, allowing flexible development of an end‐to‐end integrated high‐throughput strategy.

Production of Monoclonal Antibodies in Glycoengineered Pichia pastoris

Although improvements in antibody expression by mammalian cells are nearing maturation, efforts to improve antibody efficacy through glycoengineering are rapidly expanding. For example, the production of full length monoclonal antibodies with uniform human N-linked glycans by glycoengineered yeast has been used to optimize antibody effector function. The glycoengineered yeast expression platform not only enables elucidation of structure function relationships but also offers a robust and economically viable alternative to mammalian cell expression. This chapter provides an overview of glycobiology, engineering of P. pastoris to secrete recombinant proteins with uniform human N-linked glycans as well as bioprocess considerations in the production of full length monoclonal antibodies by a yeast based expression system.