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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.

Defining Therapeutic Window for Viral Vectors: A Statistical Framework to Improve Consistency in Assigning Product Dose Values

by Nancy Sajjadi and Janice D. Callahan
Volume 19, Open Access (Revised Aug 2023)

NOTE: Page 7 of this article has been revised to correct an error in the original article which stated that "..the probability of failing a lot when it actually is not equivalent, becomes 1-2*α." The correct equation, 1-α, has replaced the errant one and a single sentence following it was added to further clarify this concept.

ABSTRACT: Pre-clinical and clinical trials conducted to establish the minimum effective dose and the maximum tolerated dose of a viral vector assume that the assigned dose values are comparable across studies. Toxicity has been associated with high dose administration of both adenovirus and adeno-associated virus-based vectors, and increased attention must be paid to assays used to measure dose. High assay variability can be mitigated by replication and the reporting of a mean value for product lot release. The establishment of a dose specification and a testing strategy must take into account the risk of errant quality control decisions. This can be accomplished by linking assay qualification information to measurement uncertainty through a statistical framework. By adopting an equivalence approach, the risk of releasing lots with unacceptably high or low dose values is minimized by reducing measurement uncertainty. This article provides a worked-through example to introduce applicable statistical concepts and the equations necessary to facilitate their implementation in the field.

Sajjadi N, Callahan J. Defining therapeutic window for viral vectors: A statistical framework to improve consistency in assigning product dose values. BioProcess J, 2020; 19.

Originally posted online October 25, 2020. Revised article posted online August 23, 2023.


Alcohol Determination in Protein Fractionation Intermediates by Steam Distillation and Digital Refractometry

by Heinz Anderle, Matthias Spork, David Horn, Theresa Bauer, Lucia Gnauer, and Alfred Weber
Volume 19, Open Access (May 2020)

Almost 75 years after implementing the industrial ethanol fractionation process, based on the pioneering work of Edwin J. Cohn’s research group, this niche biotechnology process has not lost its importance in helping to supply patients with life-saving biotherapies. Clearly, the focus has shifted from albumin, which was first used, to the indispensable immunoglobulin preparations produced for the effective, long-term treatment of patients suffering from immunodeficiencies. In addition, the widespread and safe therapeutic use of immunoglobulins has paved the way for the development of monoclonal antibodies, now used not only for the treatment of various autoimmune diseases, but also for cancer treatment. The Cohn fractionation process, based on the different solubilities of plasma proteins, depends on the five parameters of ethanol concentration, pH, temperature, protein, and salt concentration, which are the basis of this development. Ethanol concentration can clearly be considered an essential critical parameter for this process. Therefore, it is surprising that even after the advent of process analytical technology, there is still no fast, precise, and accurate procedure at hand to determine the alcohol content of Cohn fractionation intermediates. In this paper, we will describe the implementation of an old methodology for a new purpose, which is designed to close this gap.

Anderle H, Spork M, Horn D, Bauer T, Gnauer L, Weber A. Alcohol determination in protein fractionation intermediates by steam distillation and digital refractometry. BioProcess J, 2020; 19.

Posted online May 23, 2020.

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