Manufacture Of Therapeutic Proteins Research Articles

Probable Scenarios of Process Contamination with Cutibacterium (Propionibacterium) acnes in Mammalian Cell Bioreactor.

Mammalian cell lines constitute an important part in the manufacture of therapeutic proteins. A contamination-free operation of a mammalian cell bioreactor demands constant attention to details. Cutibacterium acnes, a slow-growing bacterium, is a common skin flora and is often associated with mammalian cell bioreactor contaminations. This paper reviews the literature published about such C. acnes contaminations and presents three hypothetical contamination scenarios based on the author's experience in the hope of fostering early detection of contamination events. Bioreactor process parameters such as unusual fluctuation in dissolved oxygen, high levels of ammonia, and microscopic examination have been identified as useful tools to detect slow-growing bacteria contamination.

Arginine-enveloped virus inactivation and potential mechanisms.

Arginine synergistically inactivates enveloped viruses at a pH or temperature that does little harm to proteins, making it a desired process for therapeutic protein manufacturing. However, the mechanisms and optimal conditions for inactivation are not fully understood, and therefore, arginine viral inactivation is not used industrially. Optimal solution conditions for arginine viral inactivation found in the literature are high arginine concentrations (0.7-1 M), a time of 60 min, and a synergistic factor of high temperature (≥40°C), low pH (≤pH 4), or Tris buffer (5 mM). However, at optimal conditions full inactivation does not occur over all enveloped viruses. Enveloped viruses that are resistant to arginine often have increased protein stability or membrane stabilizing matrix proteins. Since arginine can interact with both proteins and lipids, interaction with either entity may be key to understanding the inactivation mechanism. Here, we propose three hypotheses for the mechanisms of arginine induced inactivation. Hypothesis 1 describes arginine-induced viral inactivation through inhibition of vital protein function. Hypothesis 2 describes how arginine destabilizes the viral membrane. Hypothesis 3 describes arginine forming pores in the virus membrane, accompanied by further viral damage from the synergistic factor. Once the mechanisms of arginine viral inactivation are understood, further enhancement by the addition of functional groups, charges, or additives may allow the inactivation of all enveloped viruses in mild conditions.

A LC–MS/MS platform for the identification of productivity markers in industrial mammalian cell culture media

Abstract Cell culture media formulations supplemented with soy hydrolysates for commercial scale manufacturing of therapeutic proteins were characterized by high-resolution LC–MS/MS to identify specific media components that contributed to cellular bioprocess variability. Initial untargeted screening of media formulations resulted in the combined quantification of >2500 features upon analysis by reversed-phase and hydrophilic interaction liquid chromatography. Chemometric evaluation of quantified features by Principal Component Analysis and Hierarchical Clustering revealed clear correlation between media lots and the corresponding hydrolysates implemented during formulation development stages, suggesting that the observed variation in culture performance could be influenced by components present within hydrolysates. Through multivariate data analysis, a small number of dipeptides, identified as Tyrosyl-Leucine, Phenylalanyl-Valine and LL-Cyclo(Leucylprolyl) were determined in media as potential contributors to bioprocess variability due to their statistically significant higher abundance in formulations which yielded low cellular productivity. Subsequent quantification of these features was performed through targeted LC-QQQ-MS/MS which however, revealed discrepancies between dipeptide concentration levels in media and corresponding hydrolysates presumably due to their potential instability upon interaction with other chemically defined components such as metals or chelating agents. The nutritional impact of the aforementioned features of interest on CHO cell growth performance was assessed through spiking studies in representative shake-flask cultures which however, failed to demonstrate clear correlation between dipeptide concentrations and achieved product yields further indicating the presence of a plausible synergetic effect between the identified dipeptides and alternative media components.

Evaluation of Peptide Fractionation and Native Digestion as Two Novel Sample Preparation Workflows to Improve HCP Characterization by LC-MS/MS.

Host cell proteins (HCPs) are the predominant class of impurities during manufacturing of therapeutic proteins. Previous reports have successfully shown that HCP characterization by LC-MS/MS ultimately leads to drug products of superior safety and quality. Here, we present two sample preparation strategies to approach the wide dynamic range required and compared them systematically to a standard protocol. First, we describe PreOmics fractionation as an effective 2D offline strategy. Second, we evaluate an alternative digestion approach specifically designed for purified antibodies - native (nondenaturing) digestion. Both protocols increased detection sensitivity as shown by two low level HCP models. Out of a 5 ppm spike of eight common HCPs into antibody product, all spiked proteins were positively identified. Additionally, by Universal Proteomics Standard 1 (UPS-1) spiking we obtained a comprehensive coverage of 77% below 10 ppm for the native digestion. Furthermore, we were able to detect 27% to 173% more HCPs in protein A elution pools of five different antibodies and to reject new concerns of HCP coprecipitation by pellet digestion. Although it encounters new challenges, the native digestion is very attractive due its simplicity and comparability to 2D workflows. However, for complex samples such as mock transfected cell culture fermentation, best results were obtained with peptide fractionation. This study highlights the advantages of both methods and their value to facilitate LC-MS/MS approaches to become an even more powerful tool for HCP profiling.

Effect of Iron Oxide Nanoparticles on the Oxidation and Secondary Structure of Growth Hormone

Oxidation of therapeutic proteins (TPs) can lead to changes in their pharmacokinetics, biological activity and immunogenicity. Metal impurities such as iron are known to increase oxidation of TPs, but nanoparticulate metals have unique physical and chemical properties compared to the bulk material or free metal ions. Iron oxide nanoparticles (IONPs) may originate from equipment used in the manufacturing of TPs or from needles during injection. In this study, the impact of IONPs on oxidation of a model protein, rat growth hormone (rGH), was investigated under chemical stress. Hydrogen peroxide (H2O2)- and 2,2′-azobis (2-methylpropionamidine) dihydrochloride oxidized methionine residues of rGH, but unexpectedly, oxidation was suppressed in the presence of IONPs compared to a phosphate buffer control. Fourier transform infrared spectroscopy indicated splitting of the α-helical absorbance band in the presence of IONPs, whereas circular dichroism spectra showed a reduced α-helical contribution with increasing temperature for both rGH and rGH-IONP mixtures. The results collectively indicate that IONPs can increase the chemical stability of rGH by altering the kinetics and preference of amino acid residues that are oxidized, although the changes in protein secondary structure by IONPs may lead to alterations of physical stability.

Interaction of a Macrocycle with an Aggregation-Prone Region of a Monoclonal Antibody.

Colloidal stability is among the key challenges the pharmaceutical industry faces during the production and manufacturing of protein therapeutics. Self-association and aggregation processes can not only impair therapeutic efficacy but also induce immunogenic responses in patients. Aggregation-prone regions (APRs) consisting of hydrophobic patches are commonly identified as the source for colloidal instability, and rational strategies to mitigate aggregation propensity often require genetic engineering to eliminate hydrophobic amino acid residues. Here, we investigate cucurbit[7]uril (CB[7]), a water-soluble macrocycle able to form host-guest complexes with aromatic amino acid residues, as a potential excipient to mitigate protein aggregation propensity. Two monoclonal antibodies (mAbs), one harboring an APR and one lacking an APR, were first assessed for their colloidal stability (measured as the translational diffusion coefficient) in the presence and absence of CB[7] using dynamic light scattering. Due to the presence of a tryptophan residue within the APR, we were able to monitor changes in intrinsic fluorescence in response to increasing concentrations of CB[7]. Isothermal titration calorimetry and NMR spectroscopy were then used to characterize the putative host-guest interaction. Our results suggest a stabilizing effect of CB[7] on the aggregation-prone mAb, due to the specific interaction of CB[7] with aromatic amino acid residues located within the APR. This provides a starting point for exploring CB[7] as a candidate excipient for the formulation of aggregation-prone mAbs.

Aggrescan3D (A3D) 2.0: prediction and engineering of protein solubility.

Protein aggregation is a hallmark of a growing number of human disorders and constitutes a major bottleneck in the manufacturing of therapeutic proteins. Therefore, there is a strong need of in-silico methods that can anticipate the aggregative properties of protein variants linked to disease and assist the engineering of soluble protein-based drugs. A few years ago, we developed a method for structure-based prediction of aggregation properties that takes into account the dynamic fluctuations of proteins. The method has been made available as the Aggrescan3D (A3D) web server and applied in numerous studies of protein structure-aggregation relationship. Here, we present a major update of the A3D web server to version 2.0. The new features include: extension of dynamic calculations to significantly larger and multimeric proteins, simultaneous prediction of changes in protein solubility and stability upon mutation, rapid screening for functional protein variants with improved solubility, a REST-ful service to incorporate A3D calculations in automatic pipelines, and a new, enhanced web server interface. A3D 2.0 is freely available at: http://biocomp.chem.uw.edu.pl/A3D2/

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. This paper presents an approach towards statistical process control and monitoring of protein refolding, from buffer preparation to refold quenching, during manufacturing of therapeutic proteins from Escherichia coli based systems. The proposed approach utilises measurements of online redox potential, temperature, and pH for development of a statistical model. The model has then been integrated with LabView to permit real-time monitoring of the refolding process. The proposed system has been demonstrated to successfully identify process deviations and thereby enable process control for manufacturing product of consistent quality.

High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression.

Baculovirus-based expression of proteins in insect cell cultures has emerged as a powerful technology to produce complex protein biologics for many applications ranging from multiprotein complex structural biology to manufacturing of therapeutic proteins including virus-like particles (VLPs). VLPs are protein assemblies that mimic live viruses but typically do not contain any genetic material, and therefore are safe and attractive alternatives to life attenuated or inactivated viruses for vaccination purposes. MultiBac is an advanced baculovirus expression vector system (BEVS) which consists of an engineered viral genome that can be customized for tailored applications. Here we describe the creation of a MultiBac-based VLP-factory™, based on the M1 capsid protein from influenza, and its application to produce in a parallelized fashion an array of influenza-derived VLPs containing functional mutations in influenza hemagglutinin (HA) thought to modulate the immune response elicited by the VLP.

Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity

ABSTRACTA new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.

Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.

Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20

Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins.

Construction of the Recombinant Lentiviral Vector Containing Human GH1 Gene and Its Expression in HEK293T Cells

Human growth hormone (hGH) is a protein with multiple roles in a range of biological functions such as protein, carbohydrates and lipid metabolisms as well as immunity, tissue development and overall growth. One of the major class of biopharmaceuticals in mammalian cells is the production of recombinant pharmaceutical proteins. In this study, we constructed a lentiviral vector carrying coding region of human GH1 (hGH) gene in order to production of recombinant hGH in mammalian cell line. hGH gene was amplified from a plasmid containing full-length hGH coding sequence and then cloned into the lentiviral vector pCDH-GFP. The HEK293T cells were transduced by the lentivirus particles as a targeted cell. hGH expression status in the recombinant cells were confirmed by RT-PCR. Additionally, western blotting analysis results showed that the recombinant cells maintained a stable hGH expression during five weeks of continuous culture. In conclusion, results of current study suggested that constructed lentiviral vector can potentially be used for a stable production of recombinant hGH protein in HEK293T cells. This methodology could be served as a foundation for further research and may open new insights toward therapeutic protein manufacturing.

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production.

Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture.

Yeast extract (YE), as a non-animal source additive for mammalian cell culture medium, has been widely used for manufacturing of therapeutic proteins. In the present study, one particular YE was found to have significantly improved the specific productivity (q p) of Fc-fusion protein in recombinant Chinese hamster ovary (rCHO) cell culture. In order to elucidate the intracellular effects of YE on protein productivity, steps of the target protein synthesis process were investigated to unveil their variations caused by YE addition. Stepwise analysis on Fc-fusion protein synthesis process showed that YE enhanced Fc-fusion protein gene transcription with cell cycle arrest at G1 phase; mammalian target of rapamycin (mTOR) signaling pathway was activated to enhance the translation of Fc-fusion protein, and the block in post-translational steps of Fc-fusion protein was alleviated by YE addition as well. Our results revealed the responses of multiple protein production steps to the addition of YE and provided a practical guidance for the separation and application of active compounds from hydrolysates.

Development of protein-free medium for therapeutic protein production in mammalian cells: recent advances and perspectives

Mammalian cell culture media used for the manufacture of therapeutic protein have advanced systematically from serum-containing into animal-free, protein-free and chemically defined formulations over the past decades. Initially driven by patient safety concerns associated with the use of animal-derived medium components, and later by inconsistent cell culture performance due to variability in plant-derived raw material lots, many biologics manufacturers redirect their focus on the development of proprietary media formulations and implementation of well-controlled chemically defined raw materials in all cell culture media and feeds for production processes. This article will provide an overview of current trends and objectives of industrial medium development efforts for therapeutic protein production.

N-glycome profiling of patatins from different potato species of Solanum genus.

It is hypothesized that oligosaccharides are another potential source of immunological cross-reaction between different plant allergens. Patatin is the most abundant glycoprotein in potato and has been described to have an oligosaccharide of composition Man3(Xyl)GlcNAc2(Fuc). In this work, N-glycosylation profiles of patatin proteins isolated from tubers of different potato species were investigated and compared. Oligosaccharides were released by enzymatic digestion with PNAGase A and analyzed primarily by matrix-assisted laser desorption ionization mass spectrometry. For glycan labeling, a modified version of on-target derivatization with phenylhydrazine was applied. This study found the presence of glycan structures not described previously in patatins of potato tubers, and their glycan profiles significantly differed. This knowledge about the glycosylation of potato patatins may be helpful for correct choice of potato species to decrease the presence of specific glycan epitopes causing food allergy as well as for utilization of potatoes for the manufacture of therapeutic proteins.

Process cost and facility considerations in the selection of primary cell culture clarification technology

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.

Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering

The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.

Resilient immortals, characterizing and utilizing Bax/Bak deficient Chinese hamster ovary (CHO) cells for high titer antibody production

Cell death due to apoptosis is frequently observed in large-scale manufacturing of therapeutic proteins, and can reduce product accumulation in bioreactors. Several different strategies that involve overexpression of antiapoptotic or downregulation of proapoptotic proteins have been designed in attempt to curb this problem in Chinese hamster ovary (CHO) cell culture. However, each of these designs has their own shortcomings and limits, rendering them ineffective for large-scale protein production. Recently, we have reported generation of a Bax and Bak deficient dhfr(-/-) CHO cell line using zinc-finger nucleases. Here we demonstrate that puromycin, but not methotrexate, selection can be used to generate antibody-expressing Bax and Bak deficient clones that are not only resistant to apoptosis, but that can also achieve higher titers relative to parental CHO cells due to higher cell density. Additionally, we show that Bax and Bak deficient cells have more mitochondria with healthy membrane potential, an attribute that perhaps contributes to their more potent growth compared to parental cells. Bax and Bak deficient cells do not readily apoptose, as shown by the ability to withstand high concentrations of apoptosis inducing agents, such as sodium butyrate, without a reduction in viability, growth, or titer. These traits render Bax and Bak deficient cells a potentially attractive host for production of therapeutic proteins at industrial scale.