Ion Exchange Chromatographic Methods for Purification of Therapeutic Antibodies.

Ion Exchange Chromatography has been a critical unit operation for manufacturing of therapeutic antibodies. Cation and anion exchange chromatography are used extensively to remove process-related as well as product-related impurities to obtain the final product. In this chapter, we describe the methods for separating and purifying charge variants and aggregates for manufacturing of monoclonal antibodies.

Near Infrared Spectroscopy as a PAT tool for monitoring and control of protein and excipient concentration in ultrafiltration of highly concentrated antibody formulations.

Excipient concentrations are critical quality attributes of monoclonal antibody (mAb) drug products and affect their safety and efficacy. In manufacturing processes, mAb products are formulated into the buffer containing the desired excipients using ultrafiltration (UF) and diafiltration (DF). Control of excipient concentrations is a challenge during high concentration UF due to electrostatic interactions which lead to excipient concentration drifts.

Process analytical technology application for protein PEGylation using near infrared spectroscopy: G-CSF as a case study.

Conjugation of protein therapeutics with polymers like polyethylene glycol (PEG) has been shown to increase their therapeutic efficiency. However, manufacturing of PEGylated drugs requires an additional, carefully controlled reaction step after purifying the protein, followed by further purification of over- and under-PEGylated variants. In this work, we have used a combined spectroscopic and statistical approach for monitoring and control of the PEGylation reaction for G-CSF using near infrared spectroscopy (NIRS).

Automation of Dead End Filtration: An Enabler for Continuous Processing of Biotherapeutics.

Dead end filtration is a critical unit operation that is used for primary and secondary clarification during manufacturing of both microbial and mammalian cell based biotherapeutics. Dead end filtration is conventionally done in batch mode and requires filter pre-sizing using extensive scouting studies, along with filter over-sizing before deployment to handle potential variability. However, continuous manufacturing processes require consistent use of dead-end filtration over weeks or months, with potential unpredictable variations in feed stream attributes, which is a challenge currently facing the industry.

Process Analytical Technology Implementation for Peptide Manufacturing: Cleavage Reaction of Recombinant Lethal Toxin Neutralizing Factor Concatemer as a Case Study.

The α-chymotrypsin-based cleavage reaction is necessary for manufacturing peptides using rDNA technology with tandem repeats. The current work showcases application of process analytical technology (PAT) tools for monitoring and control of this reaction, using recombinant Lethal Toxin Neutralizing Factor (rLTNF) as a case study. At-line Fourier Transform infrared spectroscopy (ATR-FTIR) combined with attenuated total internal reflectance sampling accessory was exploited to monitor the reaction.

An NIR-based PAT approach for real-time control of loading in Protein A chromatography in continuous manufacturing of monoclonal antibodies.

Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup.

High performance liquid chromatography (HPLC) based direct and simultaneous estimation of excipients in biopharmaceutical products.

Excipients are critical components of a biotherapeutic product as they have a direct impact on the product's stability and hence on its safety and efficacy. A typical biotherapeutic drug product has a variety of excipients and these need to be maintained within very tight specifications in order to ensure consistency in product quality. Hence, there is always a need for estimation of excipient concentration in the drug product.

Process analytical technology implementation for protein refolding: GCSF as a case study.

Process analytical technology is gaining interest in the biopharmaceutical industry as a means to enable consistency in processing and thereby in product quality via process control. Protein refolding is known to be significantly impacted by critical process parameters and feed material attributes including composition and pH of the solubilisation and refolding buffers. Hence, to achieve robust process control and product quality, these attributes and parameters need to be monitored.

Process for production and purification of Lethal Toxin Neutralizing Factor (LTNF) from and its economic analysis.

Background: Purification of peptides offers unique challenges with respect to obtaining the desired process yield and selectivity. Lethal Toxin Neutralizing Factor (LTNF) is a peptide that is known to neutralize snake venom in mice when the peptide is preincubated with the venom prior to intravenous injection. A process for producing highly purified recombinant LTNF has been developed.

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention.