Bioprocessing Techniques

1.51

Bioprocessing Techniques

D Cossar, University of Toronto, Toronto, ON, Canada © 2011 Elsevier B.V. All rights reserved.

1.51.1 Introduction 1.51.2 Production Strain Development 1.51.3 Fermentation Process 1.51.3.1 Inoculum Generation 1.51.3.2 Intermediate Fermentation 1.51.3.3 Production Fermentation 1.51.4 Product Recovery and Purification 1.51.4.1 Product Recovery 1.51.4.2 Product Purification 1.51.5 Process Validation 1.51.5.1 Seed Banks 1.51.5.2 Inoculum Generation 1.51.5.3 Intermediate Fermentation 1.51.5.4 Production Fermentation 1.51.5.5 Recovery and Purification 1.51.6 Process Documentation 1.51.7 Conclusion References Relevant Websites

Glossary biopharmaceutical Therapeutic product manufactured using recombinant DNA technology. bioreactor Fermentation tank with high-level process control for growth of microorganisms. current good manufacturing practices (cGMPs) Codified system of guidelines (21 CFR 11) applied in the manufacture of therapeutic products. energy spilling Process by which organisms recycle metabolites (especially ATP or NAD(P)H) using nonproductive metabolic pathways (futile cycles). laminar flow cabinet Also known as Biosafety cabinet. Enclosed system where operator and biological materials are segregated by directional flow of sterile (HEPA­ filtered) air. Reducing potential for process contamination or inadvertent exposure of operator to viable microorganisms.

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metabolic flux Flow of materials (especially nutrients) through biochemical pathways within a cell to generate energy and cell mass. operational qualification (OQ) Stage 2 of the process for using a piece of equipment in a cGMP manufacturing environment – demonstrating that the equipment functions according to specifications. Stage 1 – installation qualification (IQ) – deals with demonstration that the equipment has the specifications required. Stage 3 – performance qualification (PQ) – tests the equipment against desired process control characteristics. recombinant DNA technology Approach to generating microorganisms with desired characteristics by insertion of heterologous genetic material using plasmids (autonomous self-replicating molecules). United States Food and Drug Agency (FDA) Regulatory body responsible for controlling use of therapeutic products, including biopharmaceuticals (‘biologics’).

1.51.1 Introduction Bioprocessing is loosely defined as being the production of a value-added material from a living source. The key component in the system is that the source organism is alive and responsive to its environment. As such, the paradigm is that it will adjust its physiology to maximize efficiency in response to comparatively minor changes in its physico-chemical environment. This translates to potential variability in the nature of the system output – in our case, the product. For the bioprocess engineer, the goal is to minimize such changes in physiology by understanding and controlling the production process. The archetypical bioprocess is based on growth of a microorganism under conditions which encourage the production of a product that can be recovered at an economically viable yield and in a format which permits its use. The product is, almost by definition, of limited or no value to the producing organism. Therefore, bioprocessing can be argued to run counter to the evolutionary drive of the

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organism itself. Any reduction in wastage on the part of the production organism (either by limiting production of the product or by enhancing ability to recycle the product) will lead to increased fitness and population shift to the lower yielding variant. This is the second paradigm of bioprocessing – that the system will tend to move toward a state of lower productivity. The third paradigm of bioprocessing lies in the ability of living organisms to mutate, this being the fuel of evolution. At each round of replication, there exists the potential for DNA molecules to change (through mechanisms of misreading of template, frameshifts, recombination between sequences, gene shuffling, etc.) with consequent changes in the biochemistry of the organism. The majority of such changes are of neutral or negative impact. However, occasional mutations arise which are to the benefit of the organism in the particular environment in which it finds itself. The more challenging the environment, the more likely is a beneficial mutation. In these terms, the production environment can be considered to favor the generation of mutants. Such mutants are, by definition, stochastic and unpredictable with consequently unknown effects on the product (especially the characteristic of quality). The common element, at least in terms of operational influence, is that of environment. In any given set of physicochemical parameters, a living organism will tend to behave in a consistent manner – therefore, control of environment is a key component of bioprocessing. Unfortunately, control comes at a cost and the degree of control inevitably trades off against process economics. For example, if one considers a process raw material, the more highly characterized and tightly regulated, the more costly it will be. The more effort put into control of process physical parameters, the more costly the operation. Overall, the role of the bioprocess engineer is to strive to exert the greatest control within the economics of the final product market. A secondary consequence of this quest for control is the requirement to understand the process itself, to identify the important parameters and therefore where to apply stringent control and where a more relaxed approach can be taken. Bioprocessing is conducted using equipment which varies from the simple to the highly complex. Each piece of equipment used in the process has particular characteristics which must be considered when developing and conducting a bioprocess. This arises from the inevitable variability in operational characteristics of machines – due to design, age, and physical factors the equipment will perform in a particular manner. Thus, for example, a flask incubator will control temperature of a culture within a range around the setpoint. In a production facility, each piece of equipment is subject to operational qualification (OQ) where actual perfor­ mance is established against the specifications required for the process. Subsequent re-qualifications are conducted as a component of preventative maintenance and calibration to ensure that the equipment performs in a consistent manner. It is important for the bioprocess engineer to be aware of these operational ranges and to consider their potential impact on the bioprocess. Process development which takes account of the production scale reality is of crucial importance in bioprocessing. At bench scale, components can be added to the process without undue difficulty (including as solid). In a pharmaceutical production plant, process additions are more complicated and adding solids cannot easily be accomplished. Processing of several thousands of liters of material can take longer (or require extremely expensive equipment) than in the development lab. Mixing of cultures (which are often relatively viscous) becomes proportionately more challenging as volume increases. Use of very high purity components is trivial at bench scale but can become prohibitively expensive, if not impractical, in production. Scheduling of process steps will commonly get longer on transfer into the more rigorously regulated production environment and product almost inevitably encounters longer hold periods between stages. Such periods are also more likely to occur at higher temperatures (due to the physical difficulty in achieving low temperatures for larger volumes in holding tanks). The sensitivity of product to hold time and condition should be established as a component of process development. Bioprocessing involves a broad variety of cell types from virus to whole animals and for products which are native to the organism (and a subset of which is the organism itself) to those which are introduced by recombinant DNA technology. The detailed characteristics of the various production hosts are presented in other chapters in Volume 1 and will not be discussed here. The product itself ranges from the relatively simple organic acid (succinic, lactic, and citric) or alcohol (butanol and propanol) [1] through to complex highly bioactive proteins (erythropoietin, Granulocyte Colony Stimulating Factor (GCSF), and monoclonal antibodies). However, a generic bioprocess can be reduced to three interdependent stages – production strain development, biomass generation (product accumulation), and product recovery. The nature of these stages and the role of the bioprocess engineer within each are fundamentally different. The following sections outline the particular challenges and control techniques for these three processing compartments.

1.51.2 Production Strain Development Bioprocesses may be either based on production of native substances (i.e., those normally produced by the organism such as antibiotics and amino acids) or those introduced by recombinant DNA technology. The procedures and risks are essentially the same, just differing in emphasis. Selecting a production strain involves isolating one organism (a clone) from a heterogenous population and using this in a well-defined process to make the product. Such a clone cannot be completely and accurately described and will therefore present characteristics with the potential to surprise. Recognition of this potential and including the process flexibility to deal with it is the art of the bioprocess engineer. The development of a production strain is the base point of a bioprocess. It can also be the weakest point in the process. In general, the production strain is developed long before the decision is taken to bring a product into manufacturing. In part, this reflects the uncertainty of being able to achieve a yield of product which will support the potential market. The procedures applied to subvert the native biology of the organism to enhance yield of the desired product can be comparatively crude and have

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unforeseeable consequences for the bioprocess itself. At the very least, the bioprocess engineer should be aware of the manipula­ tions applied to prepare the production strain for these may impose restrictions or constraints on the ability to implement a controlled production. It is also relevant that, once a viable yield is obtained, the drive to market becomes the critical factor – establishing conditions where detailed characterization of the strain is not possible as a precondition to use. In other words, the bioprocess engineer must be prepared to adapt the process to the production strain. Enhancement of yield for ‘native’ products such as antibiotics or other metabolite will often involve several rounds of selection and mutation to arrive at a strain which will express a viable yield. By definition, this process runs counter to evolution of the parent organism. That is, the organism is expected to have developed the capacity to produce the substance at an appropriate level to support itself and to provide a competitive advantage over other organisms occupying an ecological niche. There are therefore several layers of metabolic process control designed to prevent a loss of overall efficiency which could arrive through wasteful production in excess of need. The archetypical example of this is in biosynthetic pathways for amino acids [2] or biofuels [3] in bacteria. These pathways are often highly branched and must be tuned to address the demands of the organism to reproduce. Thus, the organism has multiple integrated feedback loops to modulate activity of key enzymes to direct metabolic flux to supply the balance of amino acids required. Overproduction of one amino acid is therefore restricted by a negative effect of the substance on the enzymes required to make it. Dismantling these regulatory mechanisms is a prerequisite for development of a production strain for a native product. The techniques commonly applied to strain development include random mutagenesis, selection of popula­ tion outliers, and targeted gene disruption. Such procedures inevitably result in unknown collateral effects on other pathways and whose consequences may not be evident until comparatively late in process development. It is therefore the reality that the production strain for a natural product may be assumed to carry mutations which can destabilize a bioprocess. Of particular concern is the potential for the strain to revert. The overproduction of an unnecessary product puts the organism at a strong disadvantage in the presence of a competitor which produces a lower titer of the product. In consequence, the yield of the product may decline over time. In the case of non-native products which involve introduction of novel DNA sequences into the cell line, the parental cell line will carry a particular genotype which is to the benefit of the process or to its detriment. For example, the host organism may express a native protease with high affinity for the heterologous product. The selected cell line may naturally lack this activity or it may be modified through a process of selection and/or mutation. The protease negative mutant may consequentially display different behavior to the parent. It is not uncommon for recombinant microorganisms to be deliberately disabled by introduction of auxotrophic markers which would render it nonviable in the environment (thereby addressing concerns on the event of accidental release from the confines of the production plant). The requirement for exogenous supply of specific nutrients (both known and unknown) may be expected to impact on development of the final production process. In view of the inherent uncertainty involved in selecting a cell line as the basis for production, control is exercised by diligent documentation of procedures and in maintenance of a collection of intermediate versions of the organism. In this way, it is possible to conduct a risk analysis to identify potential weak points in the bioprocess and to address these during process development. For example, if reversion and yield degradation is considered likely, then the process can be prepared such that the number of generations between cell bank and harvest is minimized. Alternatively, specific components may be incorporated into the process to reduce the competitive disadvantage of the producing strain. Of particular concern in development of a cell line for production of a pharmaceutical product from a mammalian production system is for inadvertent acquisition of potentially harmful agents (e.g., prions or latent viruses) during selection. Such agents may be present in materials used for cell culture (such as media components derived from animals – sera, proteins, or their hydrolysates) [4]. Good documentation and rigorous adherence to established protocols remain the strongest assurance of freedom from unknown or unidentified infectious agents in the production cell. A broad characterization of the cell line is recommended in parallel with its use in a bioprocess. Such analysis is directed not only to reveal the mechanistic basis for product expression, but also to identify serendipitous changes from the parent strain which enhance or impede performance of the cell line. The ability to rapidly and economically sequence the entire genome of a production cell line will present a paradigm shift in characterization and safety in the bioprocessing industry. This review of the cell line is a component of continuous improvement of the process itself besides facilitating development of future processes for related products. In practice, once the production strain is determined, a system must be implemented to provide confidence that there is a stable, long-term supply of material to underpin production [5]. In the majority of cases, seed materials are stored at reduced temperature or in a dried (lyophilized) state. The principal goal is to eliminate metabolic activity and consequent change in the nature of the material over time. The conventional approach is to establish a tiered seed banking system – laying down a master cell bank (MCB) from which are derived the (manufacturing) working cell banks (WCBs) to be used for routine manufacturing.. Typically, each WCB is created by expansion of a single vial from the MCB. Such cell banks comprise the initiation point of the bioprocess and they must therefore be fully characterized through a combination of testing and manufacturing control, including protocol validation. The consequences of changing a cell bank production protocol can be extensive, especially for products which are used in a therapeutic setting. At a minimum, performance of the new banks must be validated against the historic record for previous banks. At worst, new safety trials may be required for the end product of the process. Regulatory guidelines for derivation, characterization, and maintenance of manufacturing cell banks are available from, for example, US Food and Drug Administration (FDA) [6]. The cultures for generation of a cell bank are grown in a medium and under conditions which provide consistent and homogenous growth. The procedures for preparation of cell banks are designed to maximize the competitive status of the production strain (incorporation of antibiotics as selection markers, minimizing stress, or by avoiding product expression at high level) and to ensure that raw materials are of consistent quality and availability. Cells may be harvested and resuspended in fresh

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media or the culture can be used directly. Some organisms produce spores which are ideally suited to long-term storage, but considering also that the genetic adjustment to sporulation can introduce an unacceptable degree of variability. A suitable cryopreservative (such as dimethyl sulfoxide (DMSO), skim milk, and glycerol) may be added to reduce potential of cell damage (through generation of ice crystals) on freezing [7]. The size of the various cell banks is determined by the technology available. This constrains the time between preparation of the seed material (end of culture period or addition of stabilizing agents) and filling of the last vial before sending to storage. The longer the time taken for the filling operation, the greater is the risk that the last vial filled will be different from the first. Evidently, robotic filling operations will support larger cell banks than those which rely upon manual labor. In any case, the banking process should be validated, for example, by running processes from vials taken across the fill run and demonstrating consistency of performance. The larger the seed bank, the greater the risk that extraneous microorganisms may be introduced to the material. Seed banks should be rigorously tested for the presence of contaminating organisms through the use of standard microbiological assays.

1.51.3 Fermentation Process In essence, this component of the process expands the population of the producing cell line to the volume required to meet the potential market of the product. A high volume:low cost product (such as citric acid) will require a very large batch size to be economically viable. The basis of this principle lies in the facility cost of bioprocessing. The production equipment represents a cost which can be amortized over the life span of the product, but running costs are fixed and continuous. Therefore, for any product, the costs to produce one batch are defined by the running costs of the facility (energy, personnel, etc.). These costs are distributed across the total number of units of product and, evidently, the more units made, the lower the manufacturing costs per unit and therefore the greater profitability. Over and above this factor is the stability of the product – if a significant proportion of the batch must be disposed because it will fail the quality criteria, the profit per unit sellable becomes less. Therefore, a calculation is required to match potential market (quantity of product which can be sold); price per unit sold; and shelf-life of product to manufacturing batch size. One of the most important components of bioprocessing is that of containment and separation of process materials from the external environment. Depending on the risk, more or less stringent steps are taken to prevent contamination of the process with other organisms or preventing escape of the production organism. Given the potential consequences of contamination on final product yield, a closed production system will be assumed. In this case, significant efforts are directed to directly connecting the various stages of the process. These efforts inevitably add various operational restraints on the process – for example, each transfer point must be sterilized prior to, and after, use. Thus, transfer of inoculum from a flask to a fermenter requires a connection to be made and sterilized before the fermenter culture can be started. The inoculum must be stable over the period needed to conduct these operations; otherwise, the outcome of the process can be unacceptably variable. The fermentation process routinely involves a number of stages of increasing volume and (generally) increasing complexity in a manner which permits overlap between the stages of successive processes. In this way, plant costs are spread over a number of processes to maximize efficiency through optimization of facility usage. For the purposes of illustration, a three-stage process will be outlined, comprising inoculum generation, intermediate scale fermentation, and production fermentation.

1.51.3.1

Inoculum Generation

The first stage of a bioprocess involves transfer of stored seed bank material into a nutritionally adequate growth medium. Normally, this stage is comparatively low volume to permit conservation of seed banks. The nature of microbial growth is such that the time between an initial population and a final one is independent of volume. A low seed volume in a correspondingly low culture volume will attain the same final population density (biomass per unit volume) as a high seed volume in a higher culture volume. The volume of inoculum prepared, in relation to the volume of the subsequent process stage, will define the performance characteristics of that step. Thus, a low inoculum volume will result in a longer period in the second stage but will consume fewer seed vials. It is generally preferred to bulk the number of seed vials required to inoculate each container for initiation of the process. In this way, potential variability between containers arising from dispensing of different volumes of WCB material is reduced. The seed material may be either thawed frozen sample or reconstituted lyophilized cells. In both cases, in order to maintain the integrity of the process, the transfer is conducted in a laminar flow cabinet, or similar environment where a flow of sterile air is engineered to act as a barrier to discourage concomitant entry of particles which may carry viable microorganisms. Given the difficulty in maintaining highly pure air in a large space, the size of culture vessel at this stage is normally restricted to small flasks (e.g., Fernbach or Erlenmeyer flasks, or tissue culture bottles) which can be confidently handled in a restricted space. The flasks contain an appropriate volume of media (taking into consideration effects of volumetric ratio on aeration capacity [8]) and are incubated under conditions of agitation and temperature appropriate to the production organism. After a period of time, during which the organism population increases, the culture is used to inoculate the next (larger volume) stage of the process. One of the critical issues in bioprocessing is that of development of an appropriate testing regime to establish quality of intermediates and of demonstrating consistency of the process within the capacity to control operating parameters. The operating parameters of the various bioprocess steps are defined according to their purpose. The function of the inoculum generation stage is to present a pure culture of the production strain in an appropriate state to permit rapid outgrowth in the second stage (intermediate fermentation). The two characteristics to be tested therefore are purity and final population state.

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Purity of culture is difficult to establish unequivocally due to the insensitivity of testing methods. Use of nonselective culture media and dilution plating methods will typically detect no better than about 100 organisms per milliliter of sample, based on differential colony morphology or growth characteristics. It is axiomatic that the identity of potential contaminants is unknowable with any degree of confidence. This precludes the use of highly sensitive methods such as those based on detection of specific DNA sequences of sufficient discriminatory power (identification of bacterial contamination of a mammalian culture is, however, possible using such methods). In view of this uncertainty, the bioprocess engineer is constrained to consider control of process as a means of demonstrating confidence in freedom from contamination. In this regard, the manipulations involved are carried out according to protocols which are conducted consistently and diligently recorded. Final population status is a more complex parameter to establish, having the principal characteristic of enabling a rapid and consistent establishment of active growth in the subsequent process stage. Generally, this characteristic reflects the total number of viable organisms in the sample. The factors in the flask culture which determine the viable population density include the medium used (and how it is prepared, i.e., its nutritional complement after constitution and sterilization as well as pH and buffering capacity); the physicochemical properties of the incubation system (temperature, mixing, and gas exchange); and the initial viable population in concert with the time of incubation. Experience will indicate that the medium will support a particular biomass before the organism will no longer grow, or may engender a hostile environment (inadequate buffering and production of toxic metabolites) as the culture reaches stationary phase. In batch growth, the rate of change of biomass (and therefore variation in physiology) is at its lowest absolute value toward the end of the growth phase [9]. In order to maximize the window of opportunity to achieve the desired biomass in the appropriate state, it is therefore normal to terminate the inoculum generation stage at the point at which it is entering stationary phase. The process will be conducted, on the basis of development experience, under particular conditions such that a preferred biomass is attained. The actual value at point of use may be determined by direct (e.g., mass of organisms, quantity of DNA or protein) or indirect (e.g., culture optical properties) measurement. Such methods can be relatively imprecise or require significant time to process a sample. The preferred approach is therefore to use culture time as the control variable under the knowledge that the production organism will display reproducible growth when incubated under well-controlled conditions.

1.51.3.2

Intermediate Fermentation

The intermediate fermentation is normally conducted in a well-controlled bioreactor. As for the inoculum generation step, the purpose of this stage is to produce a culture at an appropriate population density which can be transferred to the production stage in a physiological state which permits consistent outgrowth and product expression. Given the simple aims for this step, it is preferred to keep the process as simple as possible – there is, for example, no overriding need to have a very high biomass and product expression may be kept low to avoid favoring low-productivity variants. In principle, the number of generations from seed vial to final harvest point is fixed (assuming no loss of culture in the various transfers) and it is practically advantageous to have the most generations in the inoculum generation nonproduction stage which would be designed to present lowest stress and least opportunity for selection of low-yield variants. The choice of media is also dependent on the organism used but consideration should be taken to reduce the complexity of the medium at this stage. However, the intermediate fermentation can be used to condition the organism from a medium purely supporting growth to that where product is accumulated. As for the inoculum generation step, the transfer point is a target viable biomass. In the case of the intermediate fermentation, the transfer can happen relatively quickly (transfer lines are prepared ahead of use). Therefore, the concern over stability of the culture is reduced. In effect, this means that the culture can be used in mid–late log phase, rather than waiting until stationary phase as would be the preference for the inoculum. This reflects the improved control and monitoring available in the bioreactor, with consequently greater confidence in maintaining the organism in a particular physiological state at transfer. Thus, for example, pH, dissolved oxygen, or off-gas analysis can all be used to profile the process and can be applied to establish a precise and real-time transfer criterion. CO2 production rate (CPR), O2 consumption rate (OUR), or their quotient (RQ) are reliable indicators of physiological state under defined conditions and are routinely used as process control parameters. Off-line measurements of parameters (e.g., biomass by optical or gravimetric techniques) are not favored due to the complexity in removing a sample from a closed system (attach sample bottle; sterilize connections; remove sample from fermenter; resterilize connections; remove bottle with sample from fermenter – elapsed time >45 min from taking sample to being able to analyze sample). The quality criteria of the intermediate fermentation are typically purity and viable biomass. As for the inoculum generation step, it is argued that a well-controlled process will consistently achieve the target within a specified time (reflective of the averaged specific growth rate of the organism). The fermentation is monitored for changes in pH, dissolved oxygen, and exhaust gas composition (by mass spectrometry). A profile is established for the process step and future processes can be compared against this. The presence of a contaminant or a significant variant will present as shifts in the fingerprint trend of the monitored variables. It is impractical to use this fingerprint as a real-time decision parameter and it is better used for off-line analysis and establishment of confidence in demonstrating process consistency and control.

1.51.3.3

Production Fermentation

In this stage, the organism is grown under conditions designed to optimize yield of product. The culture can be grown to high biomass (assuming product expression to be directly related to cell mass) and represents a point where it is most cost-effective to implement process controls to maintain an optimum physiology. High biomass processes are normally fed-batch and require good

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control of culture pH, dissolved oxygen, and bulk mixing. These characteristics are inter-linked, especially in relation to mixing [10]. That is, both pH control and nutrient feeding imply addition of liquid to the culture; hence, dispersion of the addition as fast as possible will improve consistency. Similarly, gas transfer between air bubbles and the liquid phase is improved by mixing as well as by increasing total air:liquid surface contact area (smaller and more bubbles). The issue of aeration is among the most important of issues during scale-up from small volume reactors used for development work and the large volume production vessels. There is an inverse correlation of ability to achieve a particular gas-transfer characteristic with volume of vessel [11, 12]. The key performance criteria for this stage of the process are related to product quality and quantity. The production of a valueless (to the organism) product places the culture under the greatest stress and represents the point where any growth advantage of variants (or contaminants) will have the highest opportunity to become predominant. Where a recombinant protein is concerned, there are many promoters available which permit delaying expression (and concomitant stress) as late as possible. For native products, such a scenario is generally not possible, though there are some situations where extremes of product accumulation are avoided until a precursor is added or the culture enters a particular growth phase (e.g., production of antibiotics in stationary phase). The production stage represents the point in the fermentation process where the greatest control should be exerted. The very high metabolic demand associated with high biomass cultures in stirred-tank bioreactors predicates risk of a dramatic change in culture physiology. Of particular concern is loss of efficiency through energy spilling or futile cycles which can occur during periods of metabolic imbalance [13]. Thus, both rate of feeding and timing of feeding are crucial control parameters of production fermentation. The availability of self-tuning microprocessor controllers provides an ability to regulate feeding of a nutrient in direct response to an online measurement relating to metabolic flux – for example, CO2 production. This is especially significant against a background of increasing biomass, where specific feed rate (quantity nutrient delivered per unit biomass per unit time) is to remain constant. There are many strategies for controlling the expression of product at a particular point of the culture. In this way, product expression and growth can be effectively decoupled and biomass is accumulated before diversion of resources into product. The timing and efficiency of this switch (induction) from biomass to product are crucial to control of the process. Induction may be achieved by altering the state of a cellular regulator in a manner which permits RNA polymerase to attach to the recombinant gene and commence production of mRNA. The trigger may be addition of a specific inducer (e.g., isopropyl-β-D-thiogalactopyranoside – IPTG for the Lac promoter family), depletion of a natural repressor (e.g., phosphate for PhoA promoter), or a temperature shift (for the CI847 temperature sensitive repressor of the λ phage PL promoter). Each method has a characteristic kinetic profile with consequences for product accumulation. Inducers such as IPTG are relatively powerful – producing a rapid accumulation of mRNA – but are tempered by the organism activating exclusion mechanisms. Adding a small volume of an activator has ramifications in context of a contained process – especially relating to validation and process consistency. The use of natural control of promoter activity (e.g., phosphate depletion of limiting lactose concentration) provokes a weaker response but one which can be maintained for a longer period. In each case, there may be other mechanisms where the promoter serendipitously becomes functional – in particular, cell stress (e.g., rapid depletion of a substrate prefeeding, or onset of oxygen starvation). In such cases, the culture profile in the production fermentation can be substantially different to that of the smaller, better mixed, development fermenter. Monitoring (and control) of the production fermentation is a key issue in bioprocessing and is particularly important since direct (and online) determination of product yield is challenging. Determination of biomass via metabolic activity (CPR, OUR, and RQ) as applied to the intermediate stage is, by itself, less than adequate. Broad arrays of non-traditional (pH and dO2) sensors are being developed and it is anticipated that these will greatly improve the ability to monitor and characterize fermentation processes. Of particular relevance are those which monitor volatile components in fermentation exhaust gases [14]. Information derived from sensors which can monitor, in real time, substrate and metabolite concentrations will enable profiling of metabolic fluxes and thereby increase confidence that a process is operating according to expectation. Ultimately, a global control algorithm may be developed to optimize the production fermentation and to provide a product of consistent yield and quality, on the understanding that an organism will tend to behave consistently if the physicochemical environment is consistent. This is the motivation behind process analytical technology (PAT) initiatives [15]. Control of metabolic flux is of particular relevance where the product is a metabolite and where recovery is complicated by the presence of other, similar, metabolites. As presented in Section 1.51.2 on strain development, collateral mutations can express themselves in the production strain during the production stage of the process when conducted at scale. The inevitability of a process scale-related reduction in bulk mixing will impact on the ability of the cell to both acquire substrates and dispose of waste across the cell membrane. In practice, this will tend to a reduction in product quality (more, and potentially different, byproducts) which must be addressed by the recovery and purification steps. This predicates one of the principal axioms of process development – that the downstream processes must be designed, at small scale, to have an excess of capacity.

1.51.4 Product Recovery and Purification At this point of the bioprocess, there is a significant divergence of methodology, depending on the nature of the product. Small molecule products (chemical synthesis feedstocks, nutritional supplements, biofuels, antibiotics, vitamins, etc.) are largely recov­ ered from culture supernatant (following cell removal by centrifugation or filtration) using chemical methods (distillation, salting out, solvent extraction, ion exchange, etc.). Proteins are typically purified using chromatographic procedures (affinity, ion exchange, hydrophobic interaction, etc.) in an orthogonal series to remove unwanted proteins – either derived from the host or modified

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variants of the product. For the purposes of this review, only protein products will be discussed, though some of the principles will also apply to chemical products.

1.51.4.1

Product Recovery

The primary criterion for the recovery step is product quality. During this step, the product is exposed to an environment which is difficult to control. Assuming an intracellular product, the cells from the production fermentation are harvested from the culture medium suing either centrifugation or filtration. In each case, the time to completion of harvest is generally a significant factor compared to the small-scale development situation where the comparatively low volumes of material can be rapidly processed. During this time, the metabolic activity of the cells can be depressed (e.g., by reduction temperature) but not necessarily eliminated. It should therefore be assumed that the cell physiology is changing over the period of harvest. This can affect product quality by action of endogenous proteases or by generation of unexpected contaminating proteins which subsequently complicate the purification stages. The best that the process engineer can hope to do at this point is to establish a robust method which can be consistently conducted and which minimizes the potential for product deterioration through cell activity. Release of product from the cell is normally achieved by application of very high shear created by forcing cells through a small aperture over a high pressure drop. These processes also take time and the high shear environment under oxidizing atmospheres can combine to produce significant risk of damaging protein by denaturation, aggregation, degradation, or oxidation. Rigorous control, especially of temperature and process time should be applied to, at least, produce a product of consistent quality. The issue of oxidation is of particular concern through contact with process surfaces – the presence of corrosion on steel tanks can significantly enhance oxidation of methionine residues in proteins [16]. Following cell disruption, the product is exposed to action of cellular enzymes which may normally be compartmentalized. Adding protease inhibitors, including those which may have been developed to inhibit known components of the host cell, is a common strategy to prevent unacceptable loss of product at this point of the process. However, this also adds the burden to demonstrate their removal from the product stream during the purification steps. In some processes, particularly those involving recombinant protein expression in Escherichia coli, the protein forms insoluble aggregates in the cell (inclusion bodies). This can aid in removal of many contaminating proteins which are soluble and can be removed by centrifugation and washing. However, the technical limitations of recovering insoluble product from continuous flow centrifugation in context of a closed system (pellet is removed from the centrifuge bowl in situ by mechanical scraping) inevitably reduce process yield, and the full capacity to achieve purification is normally not realized. Following cell breakage, the product must be solubilized by addition of denaturing products (chaotropes such as urea or guanidinium hydrochloride) at a high concentration. These are normally added as solutions which are prepared to achieve the effective concentration allowing for dilution by liquid in the process stream. The use of large quantities of process raw materials can add a significant cost to the process and it is difficult to source some products at the same purity as can be accessed for small-scale development work. The final component of this process is the removal of particulate debris from the product prior to chromatographic purification. This is normally realized using tangential flow or high-capacity dead-end filtration. This step may be coupled with a concentration or buffer exchange step using ultrafiltration where product is retained by using an appropriate membrane (relating to characteristics of both composition and retention). The product is then ideally suited for application to the first column of the purification process.

1.51.4.2

Product Purification

Purification of the (protein) product is typically achieved using a series of chromatography steps with functionally distinct modes of separation. These include affinity (e.g., Protein A for antibody), ion exchange (cationic or anionic and weak or strong), hydrophobic interaction (with degrees of hydrophobicity – e.g., phenyl-, butyl-, or octyl-sepharose), and size exclusion (with variation in resolving size range). The permutations are very large when variability in resin support (e.g., sepharose, acrylic, and ceramic), linkage group (between functional group and resin support), bead size, method of elution (including salinity, pH, and polarity), and order of operations are factored. The most important process considerations are that consecutive steps should be compatible, as far as is possible, to minimize adjustments to condition the eluate from one column such that it will bind to the next. The elution strategy at bench scale is normally developed using gradient elution. However, precise reproduction of small-scale gradients in large-scale processes is difficult to achieve and it is more common to elute the product using a step change in buffer condition. The small reduction in resolving power is compensated by careful design of the overall purification strategy and by including an extra step if necessary. Wash steps are valuable to elute contaminating proteins with similar character but should take into consideration concerns of increasing scale – flow and mixing characteristics of large volume chromatography systems are such that small differences between successive elutions cannot be reliably recreated from small scale experiments. The overall cost:benefit ratio for a purification regime is highly context specific, but it is recommended that a number of alternate approaches be considered before a process is transferred to the production scale [17]. As for the other stages of the bioprocess, it is very important to understand and describe what each chromatography step achieves in terms of removal of contaminants and recovery of product. The maintenance of a closed system means that taking a sample for the purpose of deciding when to start (or stop) collecting product is not practicable. Hence, the preference is to define a real-time condition for capturing product. This is based on online analysis of the process stream (such as absorbance at a specific wavelength, pH, and conductivity), and especially

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Analysis and Control

rate of change of the measurement (corresponding to beginning or end of an eluted peak). Microprocessor-controlled chromato­ graphy skids are capable of handling a matrix of factors to fine-tune the chromatography steps. The final (polishing) step of the purification process is generally designed to get the product in a preferred solution and to be free from any remaining contaminants (including endotoxin) and especially to provide final clearance of any substances which are included in the process but which are not in the final product presentation (e.g., salts used for elution, protease inhibitors added to the cell lysate).

1.51.5 Process Validation Process validation is defined by the FDA as the collection and evaluation of data, from the process design stage throughout production, which establishes scientific evidence that a process is capable of consistently delivering quality products.(Guidance for Industry: Process validation: General principles and practices. US FDA, November, 2008 (pp. 6, l. 94–95). The most important word in this definition is scientific. Process validation can be considered as facilitating a science-based understanding of how the bioprocess works, what are the controlling variables, and where are the greatest risks in terms of product safety (quality). Validation is typically applied to each individual step, where function is defined and rigorously tested in relation to the control variables. In addition, the functionality is demonstrated through transfer to subsequent stages, not only the directly proximal step, but even up to the end of the process, depending upon a stringent analysis of potential impact or risk. The possibility of factors compounding risk must be considered and worse-case scenarios defined. Validation of a bioprocess is conducted using both small scale (using a scaled-down version of the production process) and a set of at-scale production processes. These latter processes are effectively qualification runs and control variables are not manipulated. However, additional sets of samples are normally taken for the purposes of demonstrating that the processes are consistent in terms of the performance criteria for each unit operation.

1.51.5.1

Seed Banks

When developing a testing regime for cell banks, the performance criteria for the final material (the WCB) should be defined. Typically, these would center on providing a consistent growth profile in the inoculum generation stage of the production process coupled with expected product yield capacity. The factors which control these characteristics will form the basis of testing, release for use, and validation of the cell banking processes. The outgrowth from a seed vial depends upon the viability of the bank and can be expected to relate to time of storage, storage medium, condition of cells prior to storage, and parameters of storage (temperature, rate of freezing, freezer cycling, etc.). The product expression potential will also be influenced by the mechanisms driving productivity – for example, this will be strongly affected by genetic stability. Once these characteristics are defined and tests devised to monitor them, the protocols for cell bank production may be challenged to determine failure conditions. In this regard, it may be observed that seed vial viability declines consistently with storage time. At a certain point, the viable population remaining may not be adequate to provide suitable growth of the process inoculum. Alternatively, the surviving population may show a trending bias to lower product yield (selective survival of low producing variants). Use of a culture which has been in stationary phase for a prolonged period may (or may not) show enhanced viability retention but have lower expression capacity. Concentration and quality of cryoprotectant may be important (and can vary with supplier) since these components are, by definition, cell permeable and potentially physiologically active [18, 19]. A summary of the typical control variables and potential concerns is provided in Table 1.

1.51.5.2

Inoculum Generation

The inoculum generation step consists of an expansion of the production cells from the WCB. The factors which potentially affect the outcome of this step are presented in Table 2. The performance-related criteria are purity and final culture state. In terms of purity, the manipulations required to prepare the culture containers, to introduce the WCB material, and the incubation conditions should be shown to have low risk of permitting contaminating organisms to enter the process. Validation can be done by running blank procedures where there is no seed material added but all normal operations are conducted, additionally with consideration of worst possible case (e.g., the time that the culture container is open during inoculation). The culture state relates to both outgrowth in the intermediate fermentation step and also the stability of the production characteristic. If the inoculum is too old (overgrown), the performance of the intermediate fermentation stage may be compromised. In addition, the potential for establishment of a lowproducing variant is increased. The control variable is generally time of incubation (in the absence of a readily available biomass determination) and an acceptable range is derived during development. At a minimum, the top and bottom ends of the range are tested in the manufacturing plant context. Ideally, the failure criteria should be established for the point where the functionality of the inoculum is unacceptable. As indicated above, the preference to maintain a closed system demands that the inoculum container be attached to the intermediate fermenter using a sterile union. In practice, this implies that the connection is made, the union is resterilized, and the contents of the vessel transferred. Such procedures take a minimum of 45 min (including heat-up and cool-down time). The

Bioprocessing Techniques

Table 1

687

Cell Bank production control parameters

Parameter

Variable

Comments

Growth medium

Composition

Growth conditions

Temperature, time, shear, aeration, medium composition

Harvest point

Biomass

Preparation

Time, temperature, mixing

Cryopreservative

Concentration, time

Freezing

Temperature, rate

Storage

Time, temperature

Components of the growth medium are carried forward into storage and can influence Cell Bank performance Influence growth and physiology of the organism. Acceptance criteria for the bank may include a defined biomass at a particular time. Lack of proper control of growth conditions will have impact on performance of cell banks. Will define number of vials available and physiology of organism (in relation to stage of growth at harvest). Actively growing cells are most metabolically active and would be predicted to create the highest risk of change over the course of the cell banking process. Cultures in late stationary phase may be preadapting to an inert state but may also begin to express undesirable components (involved in recycling of resources or as antibiotics). This is a critical parameter determining viability of seed material. The cells will continue to metabolize and to change between harvest and storage. This can affect viability of cell bank. Both concentration and time of exposure prior to freezing can influence cell viability and stability. Rate of freezing can have a significant impact on cell bank viability (especially for mammalian cell lines). Commercial freezers typically cycle over a range of temperatures and may periodically defrost; liquid nitrogen tanks cycle in liquid level; power outages and mechanical failure can result in the cell banks experiencing temperature fluctuations which should be modeled as part of validation exercises.

Table 2

Inoculum generation operating parameters

Parameter

Variables

Consequence

Media preparation

Will affect nutritional capacity and hence final biomass yield. May also generatce toxic or inhibitory substances

Seed bank material

Minimum mass of components (within precision of balances) Age Sterilization conditions Vessel cleaning procedures (cleaning agent residuals, inadequate rinsing) pH buffering capacity Volume Age

Incubator control

Temperature Agitation

Point of use

Age, volume, time to use

Number of viable cells will vary – changing the number of divisions (time) required to reach the target population Growth rate of the production strain is dependent on temperature and gas exchange capacity. Will affect time or ability to reach target viable biomass. Culture may be below or above target population. Viability may be decreased (if culture is old). Low productivity variants may become enriched. Total number of viable organisms transferred to subsequent stage may be higher or lower than indicated. Delay between stopping the flask culture and getting the organisms into the next stage medium can lead to reduced viability and can introduce variability in subsequent process step performance.

inoculum must be shown to be stable (retention of viability) under plant conditions (temperature and handling) over an excess of the normal time between harvesting and use. Inoculum performance should be demonstrated for times outside the normal range at both the high and low end, to establish safety margin from process failure.

1.51.5.3

Intermediate Fermentation

The intermediate fermentation offers a more highly controllable environment. However, the outcome is similar to that of the inoculum – a culture which is in an appropriate state for the efficient operation of the production stage. The point of transfer to the production vessel can be defined in terms of the growth of the organism – a target biomass within a specified time. The typical parameters which potentially influence these characteristics are presented in Table 3. During the process development phase, this potential should be evaluated and those conditions where the process fails should be identified. During process validation, the key control variables are challenged, individually or in combination, as dictated by the development experience. It is generally preferred

688

Analysis and Control

Table 3

Intermediate fermentation operating parameters

Parameter

Variable

Consequence

Media preparation

Precision of balances, transfer mechanism, sterilization conditions, cleaning agent residuals Preparation, hold time preuse, transfer system

Will affect nutritional capacity and growth rate as well as final biomass yield. Antibiotics or growth factors (amino acids, vitamins) can degrade over time, affecting culture outcome; residual volume in transfer lines leads to variable delivery of component. May affect culture performance.

Poststerilization additions pH Control Aeration and mixing

Point of use Fermenter preparation

Precision (operating range), accuracy, preparation of control solutions (acid or base) Control loop parameters (impeller rpm, air flow rate), probe (dissolved oxygen) sensitivity Time or biomass indicator (e.g., off-gas analysis) Cleaning agent residuals, change-out-of-“o”-rings or seals, product contact materials

Impeller rpm is a major contributor of shear, especially during accelerative conditions, inadequate mixing, or gas transfer can influence culture performance. Consistency of culture at point of use is a critical process parameter. Corrosion of vessel surfaces can have consequences for culture growth, grease used for o-ring seating and installation of probes can act as reservoir for contaminants (difficulty in sterilization).

to build into the process the capacity to perform appropriately at a significantly broader range. The rationale is that, even in the best-run facility, there are occasions when control parameters fail (malfunction of a probe or monitoring instrument). In such a scenario, the operating parameter can diverge from the indicated set point and this could result in a relatively expensive termination of the process. If the process failure values are established for each control variable, this can be used to permit continuation of a process if a control excursion presents itself. It is normally the case that control variables cannot be independently validated. For example, where there are two indicators of transfer point (a biomass within a target time), it is impossible to independently vary each one. The best that can be done is to test each end of the range for the operator-controlled variable – time. The physicochemical parameters are also interrelated, for example, dissolved oxygen control may involve increasing agitator rpm (and hence shear environment). Thus, these two components are inversely linked – rpm cannot be varied independently of dissolved oxygen (unless air flow is concurrently manipulated). Similarly, pH control will affect CO2 content of exhaust gas (through effects on solubility), air flow rate will modulate CO2 and O2 levels (but not CPR or OUR since the calculation includes volumetric air flow rate). In such instances, it is current practice to evaluate relative importance of each control variable in context of the biology of the organism involved. For some, shear may be of particular concern and thus agitator rpm would be given precedence over dissolved oxygen profile; for others, dissolved oxygen may be of critical concern (especially those with very high oxygen demand) and this would overrule consequences of increasing shear. The validation strategy is thus constructed around the critical process control parameters (which are inherent to the production organism). Performance of the intermediate fermentation represents the end point for validation of the inoculum generation stage. The process to expand seed vial to inoculum for the intermediate fermentation is conducted across a set of runs where the critical control parameters are varied according to worst-case considerations. These will include lowest and highest values of acceptance criteria (viable cell content) for the culture, as well as extreme ranges of other control parameters which may (in context of scientific understanding of the biology of the production organism) influence physiology – pH, temperature, or extended hold time preuse. The ability of the intermediate fermentation stage to perform according to specification in context of the defined variations in inoculum preparation represents successful completion of validation for that stage.

1.51.5.4

Production Fermentation

The key performance criteria for the production fermentation are product yield and quality. It is frequently not possible to completely assess product quality until the product has been purified; hence, surrogate performance criteria may be applied. Product yield is a function of the expression control system (point at which it is initiated) and the final yield of productive biomass. Product quality is dependent upon the physiology of the organism (i.e., the biochemical composition of the host cell). It is reasonable to claim that, if the process runs according to an established profile and the product expression mechanism is stable, then the product itself will be consistent. Validation of this step reduces, therefore, to demonstrable control of key process control parameters (as established during development) and stability of the expression mechanism. Typical variables which may affect process step outcome are presented in Table 4. The most critical process control variables are the point at which the product expression is initiated (relative to culture biomass and nutrient availability) and that at which it is terminated. In the absence of a reliable online analysis of product yield, the process is controlled on the basis of time and biomass. It can therefore be critically dependent on the quality of the input from the intermediate fermentation. The product itself will be characteristically prone to particular modification or recalcitrant contaminants (by-products). The physiology of the organism, absent major departures from the culture norms, will arguably influence the relative quantities of these components, rather than introducing completely new ones. The purification process should be designed to specifically address known variant forms of the product and the main contaminants. Identification and characterization of product forms is a critical

Bioprocessing Techniques

Table 4

689

Production fermentation operating parameters

Parameter

Variable

Consequence

Media preparation

Precision of balances, transfer mechanism, sterilization conditions, cleaning agent residuals Preparation, hold time preuse, transfer system

Will affect nutritional capacity and growth rate as well as final biomass yield. Antibiotics or growth factors (amino acids and vitamins) can degrade over time, affecting culture outcome; residual volume in transfer lines leads to variable delivery of component. May affect culture performance.

Poststerilization additions pH Control Aeration and mixing

Precision (operating range), accuracy, preparation of control solutions (acid or base) Control loop parameters (impeller rpm, air flow rate), probe (dissolved oxygen) sensitivity

Foam control

Antifoam used, criteria for addition, quantity used

Feeding regime

Feed preparation, time of feeding, rate of feed delivery Time of induction and level of inducer Time of initiation of harvest, time to completion of harvest, hold condition during harvest.

Induction conditions Harvest

Impeller rpm is a major contributor of shear, especially during accelerative conditions; inadequate mixing or gas transfer can influence culture performance. Primary concerns are adequacy of defoaming; removal of antifoam from product stream; and effects on gas transfer. Critical control parameters since supply of nutrients is a defining function for physiology. Will affect quantity and quality of product. Of particular concern in relation to quality of recovered product.

component of bioprocess development. The host organism (or processing components) will tend to generate variants of the product in response to particular characteristics of the process. Knowledge of the mechanisms of formation of byproducts or variant products is a key element in process development and design. By eliminating or at least controlling at a minimum those events will establish demonstrable process control. A significant part of bioprocess development centers on the capacity of the various purification steps to, individually and collectively, efficiently segregate these unwanted substances from the product. In the final variant of the process, there is an excess capacity at each stage to accommodate the uncontrollable natural variability of fermenta­ tion. A key demonstration is the ability of the purification step to process a worst-case fermentation (where it is predicted that there will be a maximum proportion of variant forms and contaminants relative to product) to produce a product of acceptable quality. Large-scale, fully contained, bioprocessing equipment is designed to be both clean and sterilize in place (CIP/SIP). It is a requirement to ensure that these processes are effective, especially in terms of removing residual culture components (which can contain heavily modified versions of the product) from all surfaces of the vessel following high-temperature sterilization. In addition, cleaning agents should be shown to have been cleared from the equipment before subsequent use. The burden of validation for SIP/CIP systems is substantial [20] and is frequently avoided by using disposable bag systems.

1.51.5.5

Recovery and Purification

Validation of the purification process involves tracking of product forms and contaminants across each step. When the behavior of these components is understood, then the key performance controlling variables can be defined. Thus, for example, in a chromatography step, the load and wash conditions are established to remove certain elements from the product stream. During development, there is little impediment to preparing buffers at precise values of pH and/or conductivity. In the production facility, bulk preparation of buffers involves a recipe for various component salts and will typically achieve the target composition within a range. The extent of the range depends upon accuracy of balances and the raw materials specifications (relating to purity and hydration state). The ability of each step to meet performance criteria is tested across the range which will have the most likelihood of failing to efficiently partition product from contaminant. For example, in an ion-exchange chromatography step, the wash may be designed at a particular pH and conductivity. The boundaries are established for these parameters outside which the contaminants are not effectively eluted or the product starts to come off the column. The at-scale situation is evaluated for variability in buffer preparation in relation to the failure conditions, and a risk analysis conducted to derive the target composition of the step. In addition, the purification must also be shown to effectively remove process raw materials not present in the final formulation. Typically, the latter function of the process is demonstrated using spiking tests with many times higher concentrations of the materials. Depending on the source materials, the ability of the purification step to clear bioburden (including viruses or other infectious agents) may need to be demonstrated.

1.51.6 Process Documentation During small-scale development and prevalidation, operating conditions and acceptable ranges for the various stages of the process are defined. These criteria take into consideration the production facility capability to effectively Control the process. A set of operating instructions is typically created to describe the conduct of the process; each control setpoint is provided as well as step-by­ step directions for preparation of solutions, setup of equipment, and movement of product. Accompanying these instructions is a

690

Analysis and Control

set of manufacturing records where critical process variables or operations are recorded. This process documentation is the first line in demonstrating control and reproducibility of the process. Each process variable is traceable back through validation and development to show that the range of observed values lies within a range which provides confidence that the final product has a consistent quality.

1.51.7 Conclusion The inherent variability of biological production systems (bioprocesses) represents the greatest challenge to their implementation. An understanding of the process which is obtained by careful observation of performance under different condition provides the greatest confidence of product acceptability in the market. The development of precise and accurate analytical tools to assess process performance is a crucial element in arriving at this understanding. The process is validated using a holistic approach encompassing all aspects of the process as well as the transit of product from stage to stage.

References [1] Bentley R and Bennett JW (2008) A ferment of fermentations: Reflections on the production of commodity chemicals using microorganisms. Advances in Applied Microbiology 63: 1–32. [2] Park JH and Lee SY (2010) Fermentative production of branched chain amino acids: A focus on metabolic engineering. Applied Microbiology and Biotechnology 85P: 491–506. [3] Clomburg JM and Gonzalez R (2010) Biofuel production in Escherichia coli: The role of metabolic engineering in synthetic biology. Applied Microbiology and Biotechnology 86: 419–434. [4] Hesse F and Wagner R (2000) Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. Trends in Biotechnology 18: 173–180. [5] Schiff LJ (2005) Production, characterization, and testing of banked mammalian cell substrates used to produce biological products. In Vitro Cellular and Development Biology 41: 65–70. [6] ICH Guidance (1998) Q5D: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products, (63 FR 50244; September 21, 1998). [7] Simione FP (1992) Key issues relating to the genetic stability and preservation of cells and cell banks. Journal of Parenteral Science and Technology 46(6): 226–232. [8] Vasala A, Panula J, Bollok M, et al. (2006) A new wireless system for decentralized measurement of physiological parameters from shake flasks. Microbial Cell Factories 5: 8–13. [9] Llorens JMN, Tormo A, and Martinez-Garcia E (2010) Stationary phase in Gram-negative bacteria. FEMS Microbiology Reviews 34: 476–495. [10] Soini J, Ukkonen K, and Neubauer P (2008) High cell density media for Escherichia coli are generally designed for aerobic cultivations – consequences for large-scale bioprocesses and shake flask cultures. Microbial Cell Factories 7: 26–36. [11] Zhang S, Chu J, and Zhuang Y (2004) A multi-scale study of industrial fermentation processes and their optimization. Advances in Biochemical Engineering/Biotechnology 87: 97–150. [12] Garcia-Ochoa F and Gomez E (2009) Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview. Biotechnology Advances 27: 153–176. [13] Russell JB (2007) The energy-spilling reactions of bacteria and other organisms. Journal of Molecular Microbiology and Biotechnology 13: 1–11. [14] Rudnitskaya A and Legin A (2008) Sensor systems, electronic tongues and electronic noses, for the monitoring of biotechnological processes. Journal of Industrial Microbiology and Biotechnology 35: 443–451. [15] Junker BH and Wang HY (2006) Bioprocess monitoring and computer control: Key roots of the current PAT initiative. Biotechnology and Bioengineering 95: 226–261. [16] Lam XM, Yang JY, and Cleland JL (1997) Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. Journal of Pharmaceutical Science 86(11): 1250–1255. [17] Asenjo JA and Andrews BA (2008) Protein purification using chromatography: Selection of type, modeling and optimization of operating conditions. Journal of Molecular Recognition 22: 65–76. [18] Fuller BJ (2004) Cryoprotectants: The essential antifreezes to protect life in the frozen state. CryoLetters 25(6): 375–388. [19] Pegg DE (2007) Principles of cryopreservation. Methods in Molecular Biology 368: 39–57. [20] Junker B (2001) Technical evaluation of the potential from streamlining of equipment validation for fermentation applications. Biotechnology and Bioengineering 74(1): 49–61.

Relevant Websites http://www.ema.europa.eu – European Medicines Agency; Human Medicines. http://www.fda.gov – FDA U.S. Food and Drug Administration; Vaccines, Blood & Biologics. http://www.ich.org – ICH; Welcome to Official Web Site for ICH.