Adaptation to high throughput batch chromatography enhances multivariate screening.

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored.

Development and scale-up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach.

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.

Clarification and capture of high-concentration refold pools for E. coli-based therapeutics using expanded bed adsorption chromatography.

Expanded bed adsorption (EBA) chromatography was investigated for clarification and capture of high-concentration refold pools of Escherichia coli-based therapeutics. Refolding of denatured inclusion bodies (IBs) at high protein concentration significantly improved product throughput; however, direct filtration of the refold materials became very challenging because of high content of protein precipitates formed during refolding. In addition, irreversible protein precipitation caused by high local concentration was encountered in packed bed capture during cation exchange chromatography elution, which limited column loading capacity and capture step productivity. In this study, the two issues are addressed in one unit operation by using EBA. Specifically, EBA can handle feed streams with significant amount of particles and precipitates, which eliminated the need for refold pool clarification through filtration. The relatively broad EBA elution profile is particularly suitable for proteins of low solubility and can effectively avoid product loss previously associated with on-column precipitation during capture. As the EBA resin (RHOBUST(®) FastLine SP IEX) used here has unique properties, it can be operated at high linear velocity (800-1,600 cm/h), while achieving a selectivity and impurity clearance largely comparable to the packed bed resin of the same ligand chemistry (SP Sepharose FF). Furthermore, the filtration of the EBA elution pool is easily manageable within facility capability. Overall, this study demonstrates that the EBA process helps debottleneck the purification of high-turbidity refold pools by removing precipitates and concurrently capturing the product, which can be applied to other E. coli-based therapeutics that also requires refolding of IBs.

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells.

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.