Ashraf Amanullah

Cell culture processes for monoclonal antibody production

Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

Twenty‐four well plate miniature bioreactor system as a scale‐down model for cell culture process development

Increasing the throughput and efficiency of cell culture process development has become increasingly important to rapidly screen and optimize cell culture media and process parameters. This study describes the application of a miniaturized bioreactor system as a scaled‐down model for cell culture process development using a CHO cell line expressing a recombinant protein. The microbioreactor system (M24) provides non‐invasive online monitoring and control capability for process parameters such as pH, dissolved oxygen (DO), and temperature at the individual well level. A systematic evaluation of the M24 for cell culture process applications was successfully completed. Several challenges were initially identified. These included uneven gas distribution in the wells due to system design and lot to lot variability, foaming issues caused by sparging required for active DO control, and pH control limitation under conditions of minimal dissolved CO2. A high degree of variability was found which was addressed by changes in the system design. The foaming issue was resolved by addition of anti‐foam, reduction of sparge rate, and elimination of DO control. The pH control limitation was overcome by a single manual liquid base addition. Intra‐well reproducibility, as indicated by measurements of process parameters, cell growth, metabolite profiles, protein titer, protein quality, and scale‐equivalency between the M24 and 2 L bioreactor cultures were very good. This evaluation has shown feasibility of utilizing the M24 as a scale‐down tool for cell culture application development under industrially relevant process conditions. Biotechnol. Bioeng. 2009;102: 148–160.

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process.

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl‐CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells. Biotechnol. Bioeng. 2012;109: 146–156.

Probing of C‐terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C‐terminal lysine (C‐K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C‐K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch‐to‐batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C‐K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C‐K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C‐K level for in‐process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C‐K variants were studied. The dominant effect on C‐K level was identified as the trace elements concentration. Specifically, increased C‐K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C‐K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study. Biotechnol. Bioeng. 2012;109: 2306–2315.

Feeding lactate for CHO cell culture processes: Impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO2 was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO2 were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO2 build‐up were significant. Culture growth and productivity were similar between the control and lactate‐fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO2 accumulation that could arise in certain cultures due to early depletion of lactate. Biotechnol. Bioeng. 2012; 109:1173–1186.

Automated dynamic fed-batch process and media optimization for high productivity cell culture process development.

Current industry practices for large‐scale mammalian cell cultures typically employ a standard platform fed‐batch process with fixed volume bolus feeding. Although widely used, these processes are unable to respond to actual nutrient consumption demands from the culture, which can result in accumulation of by‐products and depletion of certain nutrients. This work demonstrates the application of a fully automated cell culture control, monitoring, and data processing system to achieve significant productivity improvement via dynamic feeding and media optimization. Two distinct feeding algorithms were used to dynamically alter feed rates. The first method is based upon on‐line capacitance measurements where cultures were fed based on growth and nutrient consumption rates estimated from integrated capacitance. The second method is based upon automated glucose measurements obtained from the Nova Bioprofile FLEX® autosampler where cultures were fed to maintain a target glucose level which in turn maintained other nutrients based on a stoichiometric ratio. All of the calculations were done automatically through in‐house integration with a Delta V process control system. Through both media and feed strategy optimization, a titer increase from the original platform titer of 5 to 6.3 g/L was achieved for cell line A, and a substantial titer increase of 4 to over 9 g/L was achieved for cell line B with comparable product quality. Glucose was found to be the best feed indicator, but not all cell lines benefited from dynamic feeding and optimized feed media was critical to process improvement. Our work demonstrated that dynamic feeding has the ability to automatically adjust feed rates according to culture behavior, and that the advantage can be best realized during early and rapid process development stages where different cell lines or large changes in culture conditions might lead to dramatically different nutrient demands. Biotechnol. Bioeng. 2013; 110: 191–205.

Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media.

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu2+ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.

Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy

Dielectric spectroscopy was used to analyze typical batch and fed‐batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole–Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The β‐dispersion was analyzed using the Cole–Cole distribution parameters Δε (magnitude of the permittivity drop), fc (critical frequency), and α (Cole–Cole parameter). Furthermore, the dielectric parameters static internal conductivity (σi) and membrane capacitance per area (Cm) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAbs commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.

Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody.

Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of the sequence variant was detected by an imaged capillary isoelectric focusing (ICIEF) assay, showing a new basic species in mAb charge variant profile. The new basic variant was fractionated and enriched by ion-exchange chromatography, analyzed by reduced light and heavy chain mass determination, and characterized by HPLC-UV/MS/MS of tryptic and endoproteinase Lys-C peptide maps. A Serine to Arginine sequence variant was identified at the heavy chain 441 position (S441R), and confirmed by using synthetic peptides. The relative level of the S441R variant was estimated to be in the range of 0.3-0.6% for several mAb batches analyzed via extracted ion chromatogram (EIC). This work demonstrates the effectiveness of using integrated analytical methods to detect and identify protein heterogeneity and the importance of monitoring product quality during mAb bioprocess development.