Goals of stability evaluation throughout the vaccine life cycle

Stability studies play a critical role in assuring product quality at all points in the vaccine life cycle. At and after licensure, stability studies on quality attributes (including potency) provide a critical link between marketed and clinically evaluated vaccine product, addressing important regulatory concerns by assuring that product quality is maintained throughout the dating period. During development, stability studies are done to assure product quality and to obtain the data needed to support licensure. Stability studies may also be performed after licensure to assure that product continues to perform as it did pre-licensure, as well as to evaluate the effect on product quality of deliberately introduced manufacturing changes. At each phase in the product life cycle, it is important to consider the goals of stability evaluation and to perform appropriate statistical analyses in order to assure and reach appropriate conclusions about product quality.     

Enzyme reactions at the surface of living cells. I. Electric repulsion of charged ligands and recognition of signals from the external milieu

The dynamic behaviour of a polyelectrolyte-bound enzyme is studied when diffusion of substrate or diffusion of product is coupled to electric repulsion and to Michaelis-Menten enzyme reaction. The definition of the classical concepts of electric partition coefficients and Donnan potential of a polyelectrolyte membrane has been extended under global non-equilibrium conditions. This extension is permissible when a strong repulsion exists of substrate and product by the fixed negative charges of the membrane. Coupling between product diffusion, electric repulsion and enzyme reaction at constant advancement may result in a hysteresis loop of the partition coefficient as the product concentration is increased in the reservoir. This hysteresis loop vanishes as the rate of product diffusion increases. No hysteresis loop may occur when electric repulsion effects are coupled to substrate diffusion and reaction. The existence of multiple values of the partition coefficient for a fixed concentration of product implies that the membrane may store short-term memory of the former product concentration present in the external milieu. The occurrence of hysteresis generated by coupling enzyme reaction, product diffusion, electric partition effects at constant advancement of the reaction may be viewed as a sensing device of product concentration in the external milieu. Surprisingly, non-linearities required to generate this sensing device come from electrostatic effects and not from enzyme kinetics.     

Examining the freezing process of an intermediate bulk containing an industrially relevant protein

Numerous biopharmaceuticals are produced in recombinant microorganisms in the controlled environment of a bioreactor, a process known as Upstream Process. To minimize product loss due to physico-chemical and enzymatic degradation, the Upstream Process should be directly followed by product purification, known as Downstream Process. However, the Downstream Process can be technologically complex and time-consuming which is why Upstream and Downstream Process usually have to be decoupled temporally and spatially. Consequently, the product obtained after the Upstream Process, known as intermediate bulk, has to be stored. In those circumstances, a freezing procedure is often performed to prevent product loss. However, the freezing process itself is inseparably linked to physico-chemical changes of the intermediate bulk which may in turn damage the product.The present study analysed the behaviour of a Tris-buffered intermediate bulk containing a biopharmaceutically relevant protein during a bottle freezing process. Major damaging mechanisms, like the spatiotemporal redistribution of ion concentrations and pH, and their influence on product stability were investigated. Summarizing, we show the complex events which happen in an intermediate bulk during freezing and explain the different causes for product loss.     

Issues related to botanicals

Herbal product studies cannot be considered scientifically valid if the product tested was not authenticated and characterized in order to ensure reproducibility in the manufacturing of the product in question. Many studies refer to the use of standardized material, but in reality they are referring to chemical standardization. While chemical standardization is important, its utility is limited when the starting material is not well characterized botanically. Although the resulting studies are sound with respect to the actual product tested, adequate authentication of the product cannot be compared to other products on the market. Also, a comparison of one study to another cannot be made due to inconsistencies in the identity of the botanical matrix. The tools needed for authentication of the field plant material also depend on the plant and process involved. This could be as straightforward as botanical/morphological identification or as elaborate as genetic or chemical profiling. Authenticated raw material is the basic starting point for the development of a botanical product. However, harvesting, storing, processing and formulating methods may dramatically affect the quality and consistency of the final product by altering the desired marker components or by increasing the possibility of unwanted contaminants. Thus, validated methods to ensure quality control in manufacturing and storage are required tools for optimal efficacy and safety of the products. These controls are also critical for the evaluation of pharmacological, toxicological and clinical studies of the botanical supplements.     

Ultrastructural localization of several phosphatases with cerium

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.     

Economical factors in the assessment of various cellulosic substances as chemical and energy resources.

Economic factors in the assessment of various cellulosic substances as chemical and energy resources are many and complex. No substrate nor conversion process can be singled out as significantly advantageous. Agricultural wastes appear to have the best volume and availability characteristics. If glucose is to be the end product, then it will probably have to compete with corn syrup. If SCP is to be the end product, then productivities of 2-4 g/liter-hr must be achieved and the protein demand be such that the product can sell for at least $225/ton. If alcohol is to be the end product, then an intermediate product stream of glucose and other sugars must be obtained for 1-3cent/lb of fermentable sugars.     

Investigation of the performance of fermentation processes using a mathematical model including effects of metabolic bottleneck and toxic product on cells

A number of recent research studies have focused on theoretical and experimental investigation of a bottleneck in a metabolic reaction network. However, there is no study on how the bottleneck affects the performance of a fermentation process when a product is highly toxic and remarkably influences the growth and death of cells. The present work therefore studies the effect of bottleneck on product concentrations under different product toxicity conditions. A generalized bottleneck model in a fed-batch fermentation is constructed including both the bottleneck and the product influences on cell growth and death. The simulation result reveals that when the toxic product strongly influences the cell growth and death, the final product concentration is hardly changed even if the bottleneck is removed, whereas it is markedly changed by the degree of product toxicity. The performance of an ethanol fermentation process is also discussed as a case example to validate this result. In conclusion, when the product is highly toxic, one cannot expect a significant increase in the final product concentration even if removing the bottleneck; rather, it may be more effective to somehow protect the cells so that they can continuously produce the product.     

Influence of substrate and product diffusion on the heterogeneous kinetics of enzymic reversible reactions

When a reversible reaction is catalyzed by a surface bound enzyme, the diffusion of both substrate and product can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, the enzyme activity is decreased due to the presence of substrate and product concentration gradients in the enzyme microenvironment, and the relative kinetic importance of the two diffusion steps mainly depend on the value of a dimensionless criterion inversely proportional to the equilibrium constant. Moreover diffusional effects increase with increasing bound enzyme activity, but decrease with increasing substrate and product concentration. Analytical expressions are established for the limiting values of substrate and product concentrations in the enzyme microenvironment, as well as for the increase in half-maximal-activity substrate concentration in the presence of substrate and product diffusional limitations.     

An investigation of basic assumption in enzyme kinetics using results of the geometric theory of differential equations

Up to the present time, the following property of the product component in the reversible one substrate-one intermediate-one product enzymic mechanism has been taken only as anassumption, viz., during the course of the reaction, the time-rate of change of product concentration is never negative and the product concentration never exceeds its equilibrium value. Applying the methods of the geometric theory of ordinary differential equations it is shown that this result follows as a direct deduction from the differential equations governing the mechanism together with the initial conditions. Further, the nature of the equilibrium point as a stable node is established.     

Improving Product Specificity of Whole‐Cell Alkane Oxidation in Nonconventional Media: A Multivariate Analysis Approach

Two‐liquid‐phase reaction media have long been used in bioconversions to supply or remove hydrophobic organic reaction substrates and products to reduce inhibitory and toxic effects on biocatalysts. In case of the terminal oxyfunctionalization of linear alkanes by the AlkBGT monooxygenase the excess alkane substrate is often used as a second phase to extract the alcohol, aldehyde, and acid products. However, the selection of other carrier phases or surfactants is complex due to a large number of parameters that are involved, such as biocompatibility, substrate bioavailability, and product extraction selectivity. This study combines systematic high‐throughput screening with chemometrics to correlate physicochemical parameters of a range of cosolvents to product specificity and yield using a multivariate regression model. Partial least‐squares regression shows that the defining factor for product specificity is the solubility properties of the reaction substrate and product in the cosolvent, as measured by Hansen solubility parameters. Thus the polarity of cosolvents determines the accumulation of either alcohol or acid products. Whereas usually the acid product accumulates during the reaction, by choosing a more polar cosolvent the 1‐alcohol product can be accumulated. Especially with Tergitol as a cosolvent, a 3.2‐fold improvement in the 1‐octanol yield to 18.3 mmol L^?1 is achieved relative to the control reaction without cosolvents.     

Process Variety Modeling for Process Configuration in Mass Customization: An Approach Based on Object-Oriented Petri Nets with Changeable Structures

Mass customization strategies could be usefully deployed by companies whose products are characterized by a modular design. Typically, each module serves a specific aspect of the overall product function at varying performance levels. Each product variant (constructed through a unique combination of modular performance levels), therefore, serves to customize the overall performance of the product, thus serving the unique needs of each customer. The high demand for each module guarantees economies of scale and, eventually, low cost to customer. The rationale of configuring production processes for producing individual product variants originates from the fact that massive process data is commonly available in a firm and there exists a generic process structure underlying the production of similar products in a family. To design a decision support mechanism that constructs process configuration corresponding to a given product configuration, this paper develops a formal modeling of process variety using Petri nets. Object-oriented Petri nets (PNs) with changeable structures (OPNs-CS) are applied to deal with the issues of generic representation, constraints compliance, and operational sequence requirements. Object-oriented PN (OPN) models facilitate generic representation of product and process variety as well as their instantiations. The OPNs-CS model is tested with simulation. Based on simulation results, the optimal configuration of production processes can be determined for each individual product as well as the cohort of a product family. To illustrate the feasibility and potential of OPNs-CS based process variety modeling, a case study of process configuration for mass customized textile spindles is reported.     

In situ product recovery (ISPR) by crystallization: basic principles, design, and potential applications in whole-cell biocatalysis

The removal of inhibiting or degrading product from a bioreactor as soon as the product is formed is an important issue in industrial bioprocess development. In this review, the potential of crystallization as an in situ product removal (ISPR) technique for the biocatalytic production of crystalline compounds is discussed. The emphasis of this review is on the current status of crystalline product formation by metabolically active cells for application in fine-chemicals production. Examples of relevant biocatalytic conversions are summarized, and some basic process options are discussed. Furthermore, a case study is presented in which two conceptual process designs are compared. In one process, product formation and crystallization are integrated by applying ISPR, whereas a second, nonintegrated process is based on a known conventional process equivalent for the production of 6R-dihydro-oxoisophorone. The comparison indicates that employing ISPR leads to significant advantages over the nonintegrated case in terms of increased productivity and yield with a corresponding decrease in the number of downstream processing steps, as well as in the quantity of waste streams. This leads to an economically more interesting process alternative. Finally, a general outlook on the various research aspects of ISPR by crystallization is given.     

Assembly of the (Na+ + K+)-adenosine triphosphatase. Post-translational membrane integration of the alpha subunit

Guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates and wheat germ extracts. Antibodies to the holoenzyme (Na/K-ATPase) immunoprecipitated only a 96,000-dalton product which was identified as the alpha subunit with a molecular weight that was indistinguishable from that of mature alpha subunit. To explore the possibility that the primary translational product is integrated as such into membranes, guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates in the presence of dog pancreas microsomes; two immunoprecipitated products were detected, the 96,000-dalton alpha subunit and a 135,000-dalton new component that was integrated into the microsomal membrane since it was completely resistant to extraction with alkali. Addition of purified alpha subunit inhibited the binding of antibody to the 135,000-dalton product and extraction with urea-sodium dodecyl sulfate recovered the 96,000-dalton product, implying that the 135,000-dalton product was an alpha-chi dimer. Translation of size-fractionated poly(A+) RNA yielded evidence that the 135,000-dalton product is encoded in two separate mRNAs. The integration in vitro of the alpha subunit is, therefore, dependent on the co-translational integration into the membranes of a smaller peptide (35,000 to 40,000 daltons) which is presumably the beta subunit. Evidence was also obtained that this mechanism is present in vivo by isolation of mRNA alpha from free polysomes, as well as detection of the cytosolic form of the alpha subunit in pulse-chase experiments in MDCK cells.     

Two-phase systems: potential for in situ extraction of microalgal products

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted.Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.     

Enzyme product blot for nondestructive assay of protein catalytic function in polyacrylamide gels

A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Mechanistic mathematical model for in vivo aroma release during eating of semiliquid foods

The paper describes a mechanistic mathematical model for aroma release in the oropharynx to the nasal cavity during food consumption. The model is based on the physiology of the swallowing process and is validated with atmospheric pressure chemical ionization coupled with mass spectrometry measurements of aroma concentration in the nasal cavity of subjects eating flavored yogurt. The study is conducted on 3 aroma compounds representative for strawberry flavor (ethyl acetate, ethyl butanoate, and ethyl hexanoate) and 3 panelists. The model provides reasonably accurate time predictions of the relative aroma concentration in the nasal cavity and is able to simulate successive swallowing events as well as imperfect velopharyngeal closure. The most influent parameters are found to be the amount of the residual product in the pharynx and its contact area with the air flux, the volume of the nasal cavity, the equilibrium air/product partition coefficient of the volatile compound, the breath airflow rate, as well as the mass transfer coefficient of the aroma compound in the product, and the amount of product in the mouth. This work constitutes a first step toward computer-aided product formulation by allowing calculation of retronasal aroma intensity as a function of transfer and volatility properties of aroma compounds in food matrices and anatomophysiological characteristics of consumers.     

Circadian rhythms in Drosophila melanogaster: analysis of period as a function of gene dosage at the per (period) locus

Mutations at the per (period) locus of Drosophila melanogaster affect the period of its circadian rhythms. An analysis of published data on strains having duplications and deletions of the per locus indicates that the period is a logarithmic function of the level of the per gene product. The analysis also indicates that period is relatively insensitive to the level of that gene product; a presumed 300% increase in the gene product level produces only a 4.6% decrease in the period. The period of a strain transformed with per+ DNA conforms to the same logarithmic relationship if the level of mRNA in the transformant, which is one-tenth that in the wild type, is considered equivalent to a gene dosage one-tenth the wild type dose of 2, or 0.2. The periods of strains having various doses of mutant per alleles which shorten (pers) or lengthen (per1) the period can be fitted to the same logarithmic function. The analysis may provide an explanation for the partial dominance of pers over per+ and the dominance of per+ over per1, since it suggests that the per1 gene product is nearly inactive while the pers gene product is more than 34 times as active as the wild type product. Analysis of periods of strains heterozygous at the per locus suggests that the per gene product may be a multimeric protein. Three possible roles for the per gene product in circadian rhythmicity are discussed, including a role in synchronizing rhythm-producing cells.     

Immunogenicity of therapeutic proteins. Part 2: impact of container closures

Immunogenicity as a potential consequence of therapeutic protein administration is increasingly being scrutinized in the biopharmaceuticals industry, particularly with the imminent introduction of biosimilar products. Immunogenicity is an important safety aspect requiring rigorous investigation to fully appreciate its impact. Factors involved in product handling, such as storage temperature, light exposure, and shaking, have been implicated in immunogenicity, while container closure systems are no less important. Intended to provide a stable environment for the dosage form, container closures may also interact with a product, affecting performance and potentially enhancing immunogenicity. Glass surfaces, air-liquid interfaces, and lubricants can mediate protein denaturation, while phthalates in plastics and latex rubber are sources of extractables and leachates that may contaminate a product, causing allergic reactions and increasing immunogenicity. The manufacture of therapeutic proteins therefore requires rigorous safety evaluations not just in the context of the product, but also product containment.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.