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Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner.

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.

Cleaning-in-place (CIP) for column chromatography plays an important role in therapeutic protein production. A robust and efficient CIP procedure ensures product quality, improves column life time and reduces the cost of the purification processes, particularly for those using expensive affinity resins, such as MabSelect protein A resin. Cleaning efficiency, resin compatibility, and facility compatibility are the three major aspects to consider in CIP process design. Cleaning MabSelect resin with 50mM sodium hydroxide (NaOH) along with 1M sodium chloride is one of the most popular cleaning procedures used in biopharmaceutical industries. However, high concentration sodium chloride is a leading cause of corrosion in the stainless steel containers used in large scale manufacture. Corroded containers may potentially introduce metal contaminants into purified drug products. Therefore, it is challenging to apply this cleaning procedure into commercial manufacturing due to facility compatibility and drug safety concerns. This paper reports a safe, effective and environmental and facility-friendly cleaning procedure that is suitable for large scale affinity chromatography. An alternative salt (sodium sulfate) is used to prevent the stainless steel corrosion caused by sodium chloride. Sodium hydroxide and salt concentrations were optimized using a high throughput screening approach to achieve the best combination of facility compatibility, cleaning efficiency and resin stability. Additionally, benzyl alcohol is applied to achieve more effective microbial control. Based on the findings, the recommended optimum cleaning strategy is cleaning MabSelect resin with 25 mM NaOH, 0.25 M Na2SO4 and 1% benzyl alcohol solution every cycle, followed by a more stringent cleaning using 50 mM NaOH with 0.25 M Na2SO4 and 1% benzyl alcohol at the end of each manufacturing campaign. A resin life cycle study using the MabSelect affinity resin demonstrates that the new cleaning strategy prolongs resin life time and consistently delivers high purity drug products.

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.

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.

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant 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.