Upstream Process Parameters Research Articles

Microbial Synthesis of Lactic Acid from Cotton Stalk for Polylactic Acid Production.

Cotton stalk, a waste product in agriculture, serves as a beneficial, low-cost material as a medium for microbial synthesis of lactic acid as desired for polylactic acid synthesis. Cotton stalk was used as a substrate for microbial lactic acid synthesis, and a novel strain of Lactococcus cremoris was reported to synthesize 51.4 g/L lactic acid using cellulose recovered from the cotton stalk. In total, 18 Lactobacillus isolates were isolated from kitchen waste, soil, sugarcane waste, and raw milk samples screened for maximum lactic acid production. It was found that one of the Lactococcus cremoris isolates was found to synthesize maximum lactic acid at a concentration of 51.4 g/L lactic acid in the hydrolysate prepared from cotton stalk. The upstream process parameters included 10% inoculum size, hydrolysate containing reducing sugars 74.23 g/L, temperature 37 °C, agitation 220 rpm, production age 24 h. Only the racemic (50:50) mixture of D-LA and L-LA (i.e., D/L-LA) is produced during the chemical synthesis of lactic acid, which is undesirable for the food, beverage, pharmaceutical, and biomedical industries because only the L-form is digestible and is not suitable for biopolymer, i.e., PLA-based industry where high optically purified lactic acid is required. Furthermore, polylactic acid was synthesized through direct polycondensation methods using various catalysts such as chitosan, YSZ, and Sb2O3. PLA is biocompatible and biodegradable in nature (its blends and biocomposites), supporting a low-carbon and circular bioeconomy.

Sodium chloride impacts glycosylation and N- and O-glycan site occupancy of an Fc-fusion protein.

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.

Generic and specific recurrent neural network models: Applications for large and small scale biopharmaceutical upstream processes

The calculation of temporally varying upstream process outcomes is a challenging task. Over the last years, several parametric, semi-parametric as well as non-parametric approaches were developed to provide reliable estimates for key process parameters. We present generic and product-specific recurrent neural network (RNN) models for the computation and study of growth and metabolite-related upstream process parameters as well as their temporal evolution. Our approach can be used for the control and study of single product-specific large-scale manufacturing runs as well as generic small-scale evaluations for combined processes and products at development stage. The computational results for the product titer as well as various major upstream outcomes in addition to relevant process parameters show a high degree of accuracy when compared to experimental data and, accordingly, a reasonable predictive capability of the RNN models. The calculated values for the root-mean squared errors of prediction are significantly smaller than the experimental standard deviation for the considered process run ensembles, which highlights the broad applicability of our approach. As a specific benefit for platform processes, the generic RNN model is also used to simulate process outcomes for different temperatures in good agreement with experimental results. The high level of accuracy and the straightforward usage of the approach without sophisticated parameterization and recalibration procedures highlight the benefits of the RNN models, which can be regarded as promising alternatives to existing parametric and semi-parametric methods.

Effects of process parameters on tablet critical quality attributes in continuous direct compression: a case study of integrating data-driven statistical models and mechanistic compaction models

Continuous manufacturing of oral-dosage drug products is increasing the need for rigorous process understanding both from a process design and control perspective. The purpose of this study is to develop a methodology that analyzes the effects of upstream process parameters on continuous tablet compaction and then correlates associated upstream variables to the final tablet attributes (e.g. relative density and hardness). The impact of three process parameters (system throughput, blender speed, and compaction force) on tablet attributes is investigated using a full factorial experimental design. As expected, the compaction force was found to be the most significant process parameter. However, importantly, throughput was discovered to have a non-negligible impact which was previously unaccounted for. This impact is proposed to be related to differing levels of powder pre-compression. An empirical model for this relationship is regressed and incorporated into a flowsheet model. The flowsheet model is then used to develop an in silico design space which is compared favorably to that built from experiments. Moreover, in the future, the in silico design space based on the validated flowsheet model can provide better manufacturing flexibility and make control strategy development simpler.

Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme β-glucuronidase produced from Chinese hamster ovary cells.

β-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.25 mM butyrate addition, a temperature shift (from 37 to 32 °C), and a pH shift (from 7.0 to 6.7) along with a control (pH 7, temperature 37 °C, and no additive) on the production and quality of human recombinant β-glucuronidase (GUS) by a Chinese hamster ovary (CHO) cell line. The study was performed as two independent runs (2 bioreactors per treatment per run; n ≤ 4). Although statistically not significant, protein production slightly increased with either 0.25 mM butyrate addition (13%) or pH shift (7%), whereas temperature shift decreased production (12%, not significant). Further characterization of the purified GUS samples showed that purification selectively enriched the mannose-6-phosphate (M6P)-containing GUS protein. Noticeably, a variation observed for the critical quality attribute (CQA) of the enzyme, namely M6P content, decreased after purification, across treatment replicates and, more so, across different treatments. The dimer content in the purified samples was comparable (~25%), and no significant discrepancy was observed in terms of GUS charge variants by capillary electrophoresis analysis. MALDI-TOF/TOF analysis of released N-glycans from GUS showed a minor variation in glycoforms among the treatment groups. Temperature shift resulted in a slightly increased sialylated glycan content (21.6%) when compared to control (15.5%). These results suggest that bioreactor processes have a differential effect, and better control is required for achieving improved production of GUS enzyme in CHO cells without affecting drastically its CQAs. However, the purification method allowed for enrichment of GUS with similar CQA profiles, regardless of the upstream treatments, indicating for the first time that the effect of slight alterations in upstream process parameters on the CQA profile can be offset with an effective and robust purification method downstream to maintain drug substance uniformity.

Distribution of Antimicrobial ε-polylysine Producing Marine Microbe in Sea Water along West Coast of India

Crude glycerol is an inexpensive waste material of biodiesel manufacturing industries. The current demand is to explore useful products such as ε-Polylysine from crude glycerol (CG). In this present research, Bacillus licheniformis PL26 was found to produce 155 mg ε-Polylysine l-1 from the feed containing 2% crude glycerol and 93ml M3G medium after optimizing upstream process parameters

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett-burman design for a model monoclonal antibody.

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163-170, 2017.

468. Efficient Strategies To Deliver Reliable and High Quality Lentiviral Vector Biomanufacturing for Early Phase Clinical Trials By Optimization of Process and Regulatory Parameters

Lentiviral vectors (LVs) are important tools for gene transfer due to their efficiency and ability to stably transduce both dividing and non-dividing cells. As a result, investigators are using them as gene delivery vehicles in a wide variety of clinical applications. Nevertheless, large-scale clinical production using current good manufacturing practice (cGMP) methods comes with a set of challenges that must be considered as more clinical trials using lentiviral vectors receive regulatory approval. One important consideration in designing cGMP-compatible processes is the need to integrate regulatory considerations into manufacturing processes that are capable of producing consistent lentivirus for multiple cGMP productions. The vast majority of lentiviral vectors being used clinically have been produced by transient transfection, however, transient transfection based production is clearly labor intensive and subject to varivation. For this reason, several stable packaging cell line systems have recently been developed. While the use of these cell lines for the biomanufacturing of LV is particularly attractive for both scalability and consistency, development of such lines is time consuming and the regulatory pathway for the cGMP use of these lines has not been firmly established. With a mind toward the development of a reliable, high quality, cGMP-compliant biomanufacturing process for the clinical production of self-inactivating lentiviral vectors (SIN-LVs) we have established the CytegrityTM stable cell line system, based on the GPRG packaging cell line. Here we sought todefine key regulatory parameters to facilitate cGMP production and regulatory approval using this system. This presented a number of challenges from the establishment and tracking of clonally derived cell lines, to definition and integration of quality testing requirements, to variable responses to process operating parameters. To address these challenges, we developed approaches to optimize and facilitate stable cell line generation and selection in the context of a clear regulatory pathway for quality testing and regulatory approval. Furthermore, we established processes for material and derivative tracking allowing concurrent report generation and bioproduction assessments. Finally, we have developed mechanisms for the integration of producer cell line development, characterization, and regulatory filing with translation to at-scale cGMP-compliant bioproduction. By optimizing both regulatory and upstream process parameters, we have developed a system that allows for extensive cGMP-compliant scale-up. This “Toolbox” strategy targeting cell line generation and process conditions has allowed us to achieve desired SIN-LV productivity and quality across multiples scales and utilizing numerous different closed bioproduction systems, not only allowing production of the quantities of lentivirus vector required for Phase I and II clinical trials, but paving the way for future commercialization.

CHO starter cell lines for manufacturing of proteins with pre-defined glycoprofiles

Backround Glycosylation of protein therapeutics is influenced by a multifaceted mix of product intrinsic properties, host cell genetics and upstream process parameters. Industrial CHO cell lines may have several deficits in their glycosylation pattern for some applications, like high fucose content (corresponding to a low ADCC profile) and low galactosylation and sialylation levels (proposed to decrease activity and/ or pharmacokinetics). We have successfully applied the GlymaxX technology [1] abolishing fucose synthesis in well-established CHO DG44 and K1 platforms and pre-existing producer cell lines (glycan modulator GM1). Here we extend this strategy by other engineering approaches to enable production of protein therapeutics with desired glycosylation features. Through stable integration of other genes for glycosylation enzymes we are able to tune galactosylation (glycan modulator GM2) and sialylation (glycan modulator GM3). These glycan modulators can specifically be combined to address certain desired oligosaccharide patterns. We postulate that modulating effects of GM2 and GM3 require a specific expression level. In this case the combination of high level target protein expression and defined levels of glycan modulators becomes extremely rare. Therefore, the characterization of clones with individual stable levels of glycanmodulator expression is a prerequisite for industrial application.

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.