BioProcessing Journal

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

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.

Risk Analysis and Management Viral Vectors

adeno-associated viral vectors adenoviral vectors assay qualification dosage dose-determining assay equivalence limits therapeutic window

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

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.

Biologics Production

alcohol precipitation digital refractometry gravimetric sample dosage protein fractionation steam distillation

Porcine Serum, Trypsin, and Other Porcine-Derived Products: Swine Viruses of Importation and Adventitious Concern

The rapid and seemingly uncontrolled spread of African swine fever (ASF) throughout China and many of its neighboring countries within the last 19 months (August 2018–March 2020) has put the rest of the world on high alert. The geographic distribution of viruses of importation concern, like ASF virus (ASFV), can change very quickly, putting at risk conventional sources of porcine serum and other porcine-derived products used as ingredients in research, the manufacture of biologics, and other biomedical applications. This article reviews the 2019 information from the World Organization for Animal Health (OIE) regarding the presence or absence of eight viruses of importation concern in the swine populations of 30 countries from animal serum-producing regions of the world. Companies importing porcine raw materials for formulation into porcine products – and their customers – should be aware of the geographic location of swine diseases of importation concern. The article also identifies ten adventitious viruses of concern cited in United States Department of Agriculture (USDA) and European Union (EU) regulations that need to be tested for or eliminated through one or more barrier treatments when porcine ingredients are used in the manufacture of biologics.

Biologics Production Risk Analysis and Management

adventitious agents adventitious virus porcine serum porcine-derived products trypsin

Spent Media Analysis with an Integrated CE-MS Analyzer of Chinese Hamster Ovary Cells Grown in an Ammonia-Stressed Parallel Microbioreactor Platform

When working on biotherapeutic process development, the analysis of spent cell culture media is often a daily practice during the optimization of bioreactor conditions and media composition. The introduction of parallel microbioreactor systems has decreased the complexity and costs of process development by allowing for concurrent studies of multiple bioreactor and media variables. However, the bioreactors’ small volumes (typically less than 250 mL) limit the volume of media one can extract for daily sampling. We describe a means to analyze spent media with an integrated microchip capillary electrophoresis mass spectrometer (CE-MS) analyzer with minimal sample volume requirements and rapid analysis time. The platform was evaluated with a parallel microbioreactor system (ambr® 250) culturing a Chinese hamster ovary (CHO) cell line stressed by varying levels of ammonia (NH₃).

The spent media analysis identified net increases in the levels of the amino acids (AA) Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val in all bioreactors, with Gly levels showing increases in excess of 8-fold initial levels in all bioreactors. Other media components either steadily decreased in concentration or were completely depleted by the end of culture. For example, Asn was depleted in all of the unstressed and 10 mM NH₃-stressed bioreactors, but was approximately twice as high as the initial levels in the 30 mM NH₃-stressed bioreactors at the end of the culture periods. Also, the 30 mM NH₃-stressed condition may have caused either complete degradation or rapid consumption of choline, since it was no longer present starting at the t = 36 h sampling. Overall, the monitored media components were observed to have independent trajectories based on feeding and consumption by the cells, and depending on the stressed condition. The capability to have more frequent spent media analyses would allow for real-time observation of these process changes and associated control strategies.

Biologics Production

automation capillary electrophoresis mass spectrometer microbioreactor process control process optimization spent media analysis

Scale-Down Models to Support Process Characterization

Process characterization using qualified scale-down models (SDM) offers time and resource-saving advantages to companies developing biotherapeutics. Current approaches with glass benchtop bioreactors as SDMs have demonstrated the ability to predict process performance and product quality, but are throughput- limited by infrastructure that requires significant operational input, as well as large volumes of media and reagents. In this article, the Sartorius Stedim Biotech ambr®250 high-throughput, single-use mini bioreactor system will be discussed for its suitability as an SDM for process characterization.

Biologics Production

data integrity high-throughput process characterization scalability scale-down models single-use bioreactor

Estimating the Uncertainty of Structured Pharmaceutical Development and Manufacturing Process Execution Risks Using a Prospective Causal Risk Model (PCRM)

While many risk analysis methods describe how execution or performance risks originate and propagate through pharmaceutical and biopharmaceutical manufacturing processes and systems, few provide methods for efficiently estimating the uncertainty of an execution risk’s occurrence. This article describes prospective causal risk modeling (PCRM) for estimating the risk’s uncertainty of failures associated with executing processes, particularly when little process performance information or data is available. Building upon a basic unit of risk, the process-based system risk structure (SRS) approach is combined with PCRM to provide a method of carrying out quality risk management (QRM) exercises that properly assess both the severity and uncertainty of process execution risks. After the risks are structured using an SRS, PCRM provides a straightforward and effective method for using subjective human judgement and thought experiments to evaluate the risk process’s causal mechanisms for analyzing, evaluating, and controlling the uncertainty, including its likelihood of occurrence, of significant risks associated with developing and manufacturing pharmaceuticals. Using an SRS/PCRM-based QRM exercise, a wide variety of process execution risks can be efficiently evaluated and accepted or rejected so that important risks requiring mitigation can be identified for additional evaluation, control, and eventual acceptance.

Risk Analysis and Management

prospective causal risk modeling quality risk management system risk structure

Fetal Bovine Serum – Country of Origin, Geographic Relevance, and Labeling

With an ever-increasing number of countries involved in the collection, processing and marketing of serum, it is necessary to understand the relevance and rules relating to geographic region of origin. This article reviews and discusses the safety and quality of FBS, rules of origin, consumer market-motivated misinformation, and how mislabeled serum can be detected. The article concludes that high-quality serum needed for scientific research and biopharmaceutical products can originate from any country, as long as it is collected, imported, and processed following all the applicable regulatory and industry requirements…

Biologics Production Regulatory

fetal bovine serum geographic origin labeling quality viral safety

Fetal Bovine Serum – Geographical Origin and International Trade

It is a common belief that fetal bovine serum (FBS) collected from certain geographical regions, such as New Zealand, is of superior quality to material collected from South America. Whilst it is true that origin does have an impact on the price of serum, it does not affect the quality or biological performance of the product. FBS collected under similar conditions from any geographical region will demonstrate comparable ability to support cell growth. For FBS, the term “quality” is frequently confused with “health status.” It is the health status of the geographical region from which the serum is collected that will dictate its potential use, the availability of material for import, and eventually, the price. It should be noted that health status should be considered a result of more than just the geographical source of the material, but also the regulatory infrastructure and how well regulations are enforced by the countries within that region…

Biologics Production Risk Analysis and Management

adventitious agents fetal bovine serum geographic origin health status

Testing for Geographic Origin of Fetal Bovine Serum

Since its inception in 2006, the International Serum Industry Association (ISIA) has been focused on providing a more informative characterization standard for animal sera. A fundamental aspect of this effort has been the development of a program focused on product traceability from abattoir to end-user. This goal has been achieved in part by implementing the ISIA-sponsored audit program. Serum vendors determined to be compliant with all audit requirements are awarded ISIA Traceability Certifications. In conjunction with Oritain Global Ltd, ISIA has developed and implemented a method for establishing geographical origin of serum products. The method and its capability of determining geographical origin are described in this paper…

Regulatory

characterization chemical fingerprinting elemental analysis fetal bovine serum geographic origin product traceability standards

Gamma Irradiation of Animal Serum: General Regulatory Environment and Process Controls

This is the sixth and last in a series of articles describing and demystifying the processes involved in the gamma irradiation of serum. This serum treatment is intended to mitigate the risk of introducing adventitious contaminants into cell cultures. In this article, we discuss the regulatory environment under which gamma irradiation of serum is performed, and provide additional details on best practices for documentation of the irradiation process, selection of the contract irradiator, evaluation of risk versus benefit needed to arrive at the radiation dose range to be used, as well as an understanding of the level of remaining risk following irradiation at that dose range. Gamma irradiation should not be viewed as a means of totally eliminating risk, but rather as a means of reducing the risk of introducing adventitious agents into cell cultures. A balance must be achieved between the desire to eliminate all adventitious contaminants, and the need to retain the desired performance characteristics of the serum, once irradiated…

Regulatory Risk Analysis and Management

adventitious agents animal serum animal-derived raw materials contaminant control gamma irradiation process control

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