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Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed‐batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed‐batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:793–805, 2018 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750-757, 2015 [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are biological macromolecules with complex post-translational modifications that can be observed when assessing product variants. The N- and C-terminal heterogeneities of commercially produced antibodies have been observed and extensively studied over the past 30 years. This review summarizes the current literature on detectable antibody termini variants from cultured cells. The presence of these heterogeneities can be detected by many different analytical methods, mostly based on sequence, charge and size differences. Examples are presented that highlight terminal heterogeneities, methods of detection, and their impact on the quality of mAbs. Regulatory considerations are also discussed regarding the potential impact on product quality, safety, and efficacy. [ABSTRACT FROM AUTHOR]

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools. Published 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:745-753, 2013 [ABSTRACT FROM AUTHOR]

We surveyed 23 antibody-related marketing applications for glycoform analytical and functional information. Our database analysis shows a clear trend of increasing sophistication of analytical methods used to identify and quantify glycans. These have revealed a high degree of complexity and heterogeneity of glycans attached to antibody products. The nature of the complexity is influenced by product type and expression system, and may be associated with functional consequences in some but not all cases. [ABSTRACT FROM AUTHOR]

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50-150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ϕX174 (pI ∼ 6.7) at pH 7.5 and phage PR772 (pI ∼ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host-cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

Retrovirus-like particles (RVLPs) that are expressed during the production of monoclonal antibodies in Chinese hamster ovary (CHO) cell cultures must be removed during product recovery. Anion exchange chromatography (AEX) performed in product flow-through mode, a common component in the purification of monoclonal antibodies, has been shown to provide robust removal of a related retrovirus model, but it's ability to remove the actual RVLP impurities has not been directly investigated. We have determined the ability of a typical Q sepharose process to remove actual CHO RVLP impurities. Using small scale experiments with three model antibodies, we observe that this AEX process is capable of effectively removing both in-process and spiked RVLPs from different feedstocks containing different mAb products. In addition, we show that this AEX process also achieves a similarly high degree of RVLP removal during large scale manufacturing operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

Abstract: In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50mM NaOH/0.5M Na2SO4 with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance. [Copyright &y& Elsevier]

A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers of chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, small-scale protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6 M urea, 6 M guanidine, 100 mM NaOH or 500 mM NaOH). Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG. Under all cleaning conditions tested, the log10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles. In the two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by antibody (Ab) step yield and breakthrough. In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100 mM NaOH) or cycle 50 (500 mM NaOH). For media cycled 300+ times using 6 M urea or 6 M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident. In summary, our studies identify Ab step yield and breakthrough as performance attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled. Thus, we propose that virus removal validation studies should be performed on new media only and these attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media. [Copyright &y& Elsevier]

Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by evaluation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In a previous report, we demonstrated the utility of TaqMan fluorogenic 5′-nuclease product-enhanced reverse transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. In this report, we evaluate the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We find that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g. transmission electron microscopy, viral sequence specific TaqMan). Cell derived DNA polymerases appear to contribute only modestly to the assay background and RT activity in clarified cell culture harvests is contained largely in Type C particles. TM-PERT is linear and precise between 107and 1013 pU/ml, establishing the assay range. The assay is robust in that test article storage condition and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples. [Copyright &y& Elsevier]

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10–12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1–1000 nm) in the submicron size range. [ABSTRACT FROM AUTHOR]

Highlights • An ICP-MS methodology to determine bulk, minor and trace metal content (0.05 ppb–500 ppb) of mammalian cell culture media. • An advanced autodilution platform for autocalibration and quality control to enhance throughput. • Application of this method to demonstrate the affected homogeneity of a model therapeutic due to spikes in the concentration of copper and iron in parallel bioreactors. Abstract Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module. [ABSTRACT FROM AUTHOR]

An efficient and potentially scalable technology was evaluated to control the ice nucleation step of the freezing process for a model monoclonal antibody formulation and the effect on process performance and quality attributes of the final lyophilized product was compared with the conventional shelf ramping method of freezing. Controlled ice nucleation resulted in uniform nucleation at temperatures between −2.3 and −3.2°C while uncontrolled nucleation resulted in random nucleation at temperatures between −10 and −16.4°C. The sublimation rate (dm/dt) during primary drying was higher in the controlled nucleation cycle (0.13g/h/vial) than in the uncontrolled nucleation cycle (0.11g/h/vial). This was due to the formation of larger ice crystals, leading to lower product resistance (R p) and 19% reduction in the primary drying for the controlled nucleation cycle. Controlled ice nucleation resulted in lyophilized cakes with more acceptable appearance, no visible collapse or shrinkage and decreased reconstitution times compared with uncontrolled nucleation. There were no observed differences in the particle size, concentration (A 280nm) and presence of aggregates (A 410nm) between the two nucleation cycles when the lyophilized cakes were reconstituted. These were confirmed by SEC and protein A-HPLC analyses which showed similar peak shapes and retention times between the two cycles. However, uncontrolled nucleation resulted in cakes with larger specific surface area (0.90m2/g) than controlled nucleation (0.46m2/g). SEM images of the lyophilized cakes from uncontrolled nucleation revealed a sponge-like morphology with smaller pores while cakes from controlled nucleation cycle revealed plate-like structures with more open and larger pores. While controlled nucleation resulted in a final product with a higher residual moisture content (2.1±0.08%) than uncontrolled nucleation (1.62±0.11%), this was resolved by increasing the secondary drying temperature. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to use QbD approaches to evaluate the effect of several variables and their interactions on quality of a challenging model murine IgG3κ monoclonal antibody (mAb), and then to obtain an optimized formulation with predefined quality target product profile. This antibody was chosen because it has a propensity to precipitate and thus represents a challenge condition for formulation development. Preliminary experiments were conducted to rule out incompatible buffer systems for the mAb product quality. A fractional factorial experimental design was then applied to screen the effects of buffer type, pH and excipients such as sucrose, sodium chloride (NaCl), lactic acid and Polysorbate 20 on glass transition temperature (), monoclonal antibody concentration (A 280), presence of aggregation, unfolding transition temperature (T m) of the lyophilized product, and particle size of the reconstituted product. A Box–Behnken experimental design was subsequently applied to study the main, interaction, and quadratic effects of these variables on the responses. Pareto ranking analyses showed that the three most important factors affecting the selected responses for this particular antibody were pH, NaCl, and Polysorbate 20. The presence of curvature in the variables’ effects on responses indicated interactions. Based on the constraints set on the responses, a design space was identified for this mAb and confirmed with experiments at three different levels of the variables within the design space. The model indicated a combination of high pH (8) and NaCl (50mM) levels, and a low Polysorbate 20 (0.008mM) level at which an optimal formulation of the mAb could be achieved. Moisture contents and other analytical procedures such as size exclusion chromatography, protein A analysis and SDS-PAGE of the pre-lyophilized and final reconstituted lyophilized products indicated an intact protein structure with minimal aggregation after formulation and lyophilization. In conclusion, experimental design approach was effective in identifying optimal concentrations of excipients and pH for this challenging monoclonal antibody formulation. [Copyright &y& Elsevier]

Abstract: Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log10 clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6log10 dynamic range with a limit of detection (LOD) of ≈1 genome copy/μL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies. [Copyright &y& Elsevier]

Abstract: The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to naïve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age. [Copyright &y& Elsevier]

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small ‘naked’ viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development. [Copyright &y& Elsevier]

Abstract: We evaluated viral clearance in multiply-cycled anion-exchange media run in flow-through mode. We found that anion-exchange columns do not lose viral clearance capacity after extensive re-use, if they are cleaned with recommended buffers that do not chemically degrade the media. In contrast, anion-exchange (AEX) columns that are not cleaned or are cleaned with buffers that chemically degrade the media lost viral clearance capacity after extended use. In these cases, other performance attributes that changed at the same time were increased band spreading, decreased DNA clearance and accumulating backpressure that prevented re-use past 80–120 cycles. Thus, our data suggests that flow through mode anion-exchange columns that are cleaned with recommended cleaning buffers, and periodically monitored for band spreading, DNA clearance and/or backpressure need not be re-evaluated for viral clearance at the end of the validated media lifetime. [Copyright &y& Elsevier]