Mili Pathak

Continuous Processing for Production of Biopharmaceuticals

The merits of continuous processing over batch processing are well known in the manufacturing industry. Continuous operation results in shorter process times due to omission of hold steps, higher productivity due to reduced shutdown costs, and lowers labor requirement. Over the past decade, there has been an increasing interest in continuous processing within the bioprocessing community, specifically those involved in production of biotherapeutics. Continuous operations in upstream processing (perfusion) have been performed for decades. However, recent development of continuous downstream operations has led the industry to envisage an integrated bioprocessing platform for efficient production. The regulators, key players in the biotherapeutic industry, have also expressed their interest and willingness in this migration from the traditional batch processing. This paper aims to review major developments in continuous bioprocessing in the past decade. A discussion of pros and cons of the different proposed approaches has also been presented.

Fermentanomics: Relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real‐time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration‐related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.

Mechanistic understanding of fouling of protein A chromatography resin

This paper aims to provide a thorough understanding of how fouling of Protein A resin takes place. Binding and mass transport properties of widely used agarose-based Protein A resin, MabSelect SuRe™, have been examined to understand the mechanism of resin fouling. There could be various factors that impact resin fouling. These include product/impurity build-up due to components in the feed material and ligand degradation due to the use of harsh buffers. To unravel their contributions, cycling studies were performed with and without product loading. The results presented in this paper provide a lucid understanding of the causative factors that limit Protein A chromatographic resin lifetime. The capacity fall for protein A resin at the end of 100th cycle due to use of feed material was found to be five times greater than that without using feed material. Compared to the fresh resin, the cycled resin samples shows 24% reduction in particle porosity and 51% reduction in pore mass transfer coefficient. Transmission electron microscopy (TEM) was used to qualitatively monitor accumulation of foulants on the cycled resin. Fouled resin sample contained a dense residue in the interior and exterior of resin particle both as a film at the bead surface and as granules. The surface activation energy increased five times in the case of fouled resin sample. The major event in fouling was identified as the non-specific adsorption of the feed material components on resin, signaling that pore diffusion is the rate limiting step. It is anticipated that these findings will assist in development of a more robust and economical downstream manufacturing process for monoclonal antibody purification.

Analytical QbD: Development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates

This paper presents a quality by design (QbD) based development of a novel native PAGE (N‐PAGE) method as a low‐cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N‐PAGE relevant to those academicians and other small companies involved in early‐stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N‐PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD‐based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N‐PAGE has been in use for protein analysis for several decades, use of N‐PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.

Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics

Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G‐CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP‐HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development.

Analytical characterization of in vitro refolding in the quality by design paradigm: Refolding of recombinant human granulocyte colony stimulating factor

Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process as per the expectations laid out in the Quality by Design paradigm.

Fluorescence based real time monitoring of fouling in process chromatography

A real time monitoring of fouling in liquid chromatography has been presented. The versatility of the approach has been proven by successful implementation in three case studies with an error <1%. The first application demonstrates the monitoring of protein A ligand density and foulant concentration for assessing performance of protein A chromatography resin during purification of monoclonal antibodies. The observations have been supported from LC-MS/MS studies that were independently performed. The second application involves monitoring of foulant deposition during multimode cation exchange chromatography based purification of human serum albumin. Finally, in the third application, monitoring of foulants during multimodal hydrophobic interaction chromatography of recombinant human granulocyte colony stimulating factor is demonstrated. In all three cases, it is observed that the fluorescence intensity consistently increases with resin reuse as more foulants are deposited over time. The proposed approach can be readily used for real time monitoring of fouling and process control.

Monitoring Quality of Biotherapeutic Products Using Multivariate Data Analysis

Monitoring the quality of pharmaceutical products is a global challenge, heightened by the implications of letting subquality drugs come to the market on public safety. Regulatory agencies do their due diligence at the time of approval as per their prescribed regulations. However, product quality needs to be monitored post-approval as well to ensure patient safety throughout the product life cycle. This is particularly complicated for biotechnology-based therapeutics where seemingly minor changes in process and/or raw material attributes have been shown to have a significant effect on clinical safety and efficacy of the product. This article provides a perspective on the topic of monitoring the quality of biotech therapeutics. In the backdrop of challenges faced by the regulatory agencies, the potential use of multivariate data analysis as a tool for effective monitoring has been proposed. Case studies using data from several insulin biosimilars have been used to illustrate the key concepts.

Protein A chromatography resin lifetime—impact of feed composition

Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose‐based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC‐MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding.

Development of a low-cost, high-throughput native polyacrylamide gel electrophoresis (N-PAGE) protocol for lipoprotein sub-fractionation using Quality by Design approach.

Ratio of low density to high density lipoprotein concentration is critical for normal functioning of human body. Deviation in this ratio has been linked to various diseases, many of which are fatal if not diagnosed at early stages. For example, cardiovascular diseases (CVD) have been linked to the level of low density lipoprotein (LDL). Henceforth, detection of the lipoprotein subtractions is crucial for health of an individual. To date, methods like ultracentrifugation, nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and gradient gel electrophoresis (GGE) have been used for separation and identification of lipoprotein types and subtypes. However, these methods are expensive, time consuming and require specialized equipments and expertise. This paper aims to propose a low-cost, high-throughput native polyacrylamide gel electrophoresis (N-PAGE) based protocol for analysis of lipoproteins. Quality by Design (QbD) based approach has been utilized. The initial screening of parameters was followed by a fractional factorial design to optimize the protocol. The lipoprotein subtractions obtained by the optimized protocol were compared with the commercially available and commonly used Lipoprint(®) Lipoprotein Subfractions Testing System from Quantimetrix. The proposed method gave comparable results to those obtained with the commercial system. The proposed method is capable of analysis of up to forty different samples in two hours at a cost of approximately 2