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A Novel, Risk-based Approach for Predicting the Optimum Set of Process and Cell Culture Parameters for Scaling Upstream Bioprocessing

By Adrian Stacey, Jochen Scholz, and Sinyee Yau-Rose
Volume 20, Open Access (September 2021)

The ability to scale a cell culture effectively and efficiently, from lab to manufacturing, is critical to maximizing productivity whilst minimizing the risk of run failures and delays that can cost millions of dollars per month. The task of scaling well, however, is still considered to be a challenge by many upstream scientists, and this can be an exercise in trial and error. Traditionally, scaling has most often been performed using arithmetic in a spreadsheet and/or simple “back of an envelope” calculations. For some, it may even come in the form of support from a team of data scientists using advanced analytical software. This dependency on what some consider to be complex mathematics or statistics has resulted in the common consideration of using just one scaling parameter at a time, one scale at a time.

However, it is difficult to determine easily or optimally, from the start, whether a process successfully transfers across scales based on only one process parameter, at one scale. In this article, we describe the benefits of using a risk-based approach to scaling, and the development of a software scaling tool known as BioPAT® Process Insights for predictive scale conversion across different bioreactor scales. BioPAT Process Insights can be used to consider multiple parameters and across multiple scales simultaneously, from the start of a scaling workflow. We briefly describe how it was used in a proof-of-concept scale-up study to allow a faster, more cost-effective process transfer from 250 mL to 2000 L. In summary, using BioPAT Process Insights, in conjunction with a bioreactor range that has comparable geometry and physical similarities across scales, has the potential to help biopharma manufacturing facilities reach 2000 L production-scale volumes with fewer process transfer steps, saving both time and money during scale-up of biologics and vaccines.

Stacey A, Scholz J, Yau-Rose S. A novel, risk-based approach for predicting the optimum set of process and cell culture parameters for scaling upstream bioprocessing. BioProcess J, 2021; 20.

Posted online September 28, 2021.


Monoclonal and Polyclonal Antibodies as Biological Reagents for SARS-CoV-2 Diagnosis Through Nucleocapsid Protein Detection

by Daily Hernández, Cristina García, Marcos González, Hilda Garay, David Diago, Luis Guzmán, Williams Ferro, Mayté Quintana, Leonardo Gómez, Bárbara Chávez, Virginia Capó, Hasel Aragón, Amalia Hernández, Samy Puertas, Pedro Puente, Regla Somoza, Grechen Menéndez, Sigifredo Padilla, Israel Borrajero, and Rodolfo Valdés
Volume 20, Open Access (June 2021)

SARS-CoV-2 is an enveloped, positive-strand RNA virus that contains four structural proteins: spike, envelope, membrane, and nucleocapsid (N-protein). The N-protein participates in virus RNA packaging and particle release, is conserved within SARS-CoV-2 isolates, is highly immunogenic, and is abundantly expressed during SARS-CoV-2 infection. For these reasons, the N-protein could be used as a marker for detecting SARS-CoV-2 in early infection when antibodies against SARS-CoV-2 have not been produced yet. This paper describes the production and characterization of mouse monoclonal antibodies (mAb) and rabbit polyclonal antibodies (pAb) specific for the M20P19 peptide (N-protein linear epitope) for detection purposes. For this study, B-cell hybridomas were generated from mice independently immunized with two different M20P19 peptide-carrier protein conjugates: (1) meningococcal protein P64K; and (2) the keyhole limpet hemocyanin (KLH). Rabbits were also independently immunized with these two immunogens. Study results demonstrated that the M20P19 peptide was very immunogenic in mice and rabbits, and both mAb and pAb specifically recognized the non-conjugated M20P19 peptide, conjugated M20P19 peptide, and N-protein with high affinity and specificity, which could allow SARS-CoV-2 detection by different analytical techniques. This study corroborated that specific and high affinity constant mAb and pAb against the M20P19 peptide can be used as biological reagents for specific and rapid SARS-CoV-2 detection, mainly in tissue samples.

Hernández D et al. Monoclonal and polyclonal antibodies as biological reagents for SARS-CoV-2 diagnosis through nucleocapsid protein detection. BioProcess J, 2021; 20.

Posted online June 23, 2021.


Demonstrating the Equivalence of Traditional Versus Automated Buffer Preparation Methods Using In-Line Conditioning Control Modes to Manage Incoming Stock Solution Variability

by Karolina Busson, Robbie Kamperveen, and Enrique Carredano
Volume 20, Open Access (Apr 2021)

In-line conditioning (IC) is a form of dilution where a process buffer is formulated in-line from concentrated stock solutions of acids, bases, and salts that are mixed with the correct amount of water-for injection (WFI). This new buffer preparation strategy must prove its equivalency to buffers made the traditional way (i.e., weighing salts, stirring in water, titrating with acid or base). In this paper, such a demonstration is presented using two control modes: (1) ratio control with flow feedback; and (2) pH/conductivity feedback. To obtain the necessary parameters for an error propagation analysis, a robustness study has been performed. Our analysis showed that with low incoming variability, or when the uncertainty of the stock solutions is below 2%, the two modes of control give comparable performance. When the uncertainty increases, so does the uncertainty of ratio control with flow feedback, more with respect to conductivity than pH, while the precision of pH/conductivity feedback remains at the same level. The choice of control should therefore take into consideration the critical process parameters, their tolerances, and the input variability in the stock solution concentration. In situations where there are higher variabilities in stock solution concentrations or process temperatures, this study suggests that pH/conductivity feedback might be a better option.

Busson K, Kamperveen R, Carredano E. Demonstrating the equivalence of traditional versus automated buffer preparation methods using in-line conditioning control codes to manage incoming stock solution variability. BioProcess J, 2021; 20.

Posted online April 27, 2021.


Seed Train Process Intensification Strategy Offers Potential for Rapid, Cost-Effective Scale-Up of Biosimilars Manufacturing

by Rajib Malla, Dhaval D. Shah, Chinmay Gajendragadkar, Vijayalakshmi Vamanan, Deepak Singh, Suraj Gupta, Deepak Vengovan, Ravi Trivedi, Henry Weichert, Melisa Carpio, and Krishna Chandran
Volume 20, Open Access (Apr 2021)

A perfusion approach at N-1, where cells stay in the exponential growth phase throughout the entire culture duration, is becoming more common as a strategy for process intensification. This is because the higher cell densities it generates allows manufacturers to skip seed stages and reduce process transfer time through multiple bioreactor sizes, thus providing more cost-effective biologics production in smaller facilities. However, this N-1 perfusion approach requires optimization. In this article, we describe the development and proof-of-concept studies with single-use rocking motion perfusion bioreactors in which we have achieved a ten-fold increase in viable cell count in N-1 seed stage, compared to the fed-batch control process, in just 6–8 days. We also mention in detail how we inoculated a 50 L bioreactor production run using this intensified seed train and show comparable growth kinetics and yield with a control process, also at 50 L scale. Using this intensification approach in the future will help our manufacturing facility, the Biopharma Division of Intas Pharmaceuticals Ltd., reach 4000 L production-scale volumes with fewer process transfer steps, and without changing the feeding strategy or production bioreactors of our biologics’ portfolio.

Malla R et al. Seed train process intensification strategy offers potential for rapid, cost-effective scale-up of biosimilars manufacturing. BioProcess J, 2021; 20.

Posted online April 23, 2021.


Design and Performance of Viral Clearance Studies with Tissue-Derived Products

by Stephen Stoltzfus and Katherine F. Bergmann
Volume 20, Open Access (Feb 2021)

Tissue-derived products are a class of biological materials harvested directly from animal or human tissue, in contrast to recombinant DNA materials grown in cell culture bioreactors. Tissue-derived products are often used for structural purposes and are typically regulated as medical devices. However, when used to treat human patients, tissue-derived products are subject to many of the same concerns as recombinant DNA biotherapeutics, with viral safety being one of them. To address this, the tissue source material must undergo a risk analysis and testing regimen for the presence of viral contaminants. In addition, viral clearance studies must be performed to evaluate whether the purification process is robust enough to remove and/or inactivate viruses that may be present in the starting material.
The goals of viral clearance studies are the same for tissue-derived products and biotherapeutics, but the design and performance of these studies can be quite different because of the diverse nature of the materials. In this article, we will present an overview of viral clearance studies for tissue-derived products based on our experience in performing a large number of such studies. Rather than discussing the issues related to viral clearance in general, our focus will be on the unique challenges that tissue-derived products pose.

Stoltzfus S, Bergmann KF. Design and performance of viral clearance studies with tissue-derived products. BioProcess J, 2021; 20.

Posted online February 5, 2021.


A Direct Method to Monitor Glutathione Stability in High Concentration Protein Formulations

by Seth Keever, Bassam Nakhle, and Bernice Yeung
Volume 20, Open Access (Jan 2021)

Due to its antioxidant properties and favorable safety profile, glutathione (GSH) finds use in protein formulations by improving overall protein stability. Once degraded, primarily by oxidation into glutathione disulfide (GSSG), the protecting effect of GSH is lost. A simple, direct method using reversed-phase separation and charged-aerosol detection (RP-CAD) to quantitate GSH is described in this paper. The analytical methodology is also capable of monitoring several by-product degradants of GSH, both oxidative and non-oxidative. For high-concentration protein formulations, the method provides direct analysis of GSH and its degradants in the presence of protein at up to 225 mg/mL simply through a dilution of the sample. Quantitation of many amino acids typically included in pharmaceutical protein formulations is also possible. Use of an online diverting valve in the method prevents interference in the detector from the high protein concentration in formulation. Accuracy and effectiveness of this method is demonstrated through monitoring the stability of GSH in high-concentration protein formulations through confirmation of GSH concentration and mass-balance of its loss over time. Monitoring GSH stability in protein formulations is necessary, as GSH concentration is indicative of protein stability.

Keever S, Nakhle B, Yeung B. A direct method to monitor glutathione stability in high concentration protein formulations. BioProcess J, 2021; 20.

Posted online January 12, 2021.

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