BY PHASE


Steps on Drug Production for Large Molecules
  • Drug Discovery

    Searching for a potential molecule to become a new drug.
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  • Pre-Clinical Trial

    Utilizing laboratory and animal testing to determine if the drug is safe for human testing.
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  • Clinical Trial Approval

    Applying for Investigational New Drug (IND) to ensure safety of clinical trial.
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  • Clinical Trials

    Determining the safety and efficacy of the drug on clinical trial volunteers.
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  • Marketing Approval

    Applying for New Drug Application (NDA) or biologics license application for marketing .
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  • Post-Marketing Surveillance

    Monitoring the long term effects of the drug on a greater population.
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Drug Discovery

Drug Discovery
Lead Identification
Lead Optimization
Lead Development
The choice of antibody platform, appropriate in vivo and in vitro standards and the pharmacodynamics and pharmacokinetic models should have been first considered.
It utilizes natural, synthetic and /or semi synthetic sources for potential selection of candidate molecule.
Structure-Activity Relationships are performed to improve potency and selectivity of the candidate molecule.
Improvement of biochemical biophysical, physicochemical and druglike properties like expression, solubility, stability, aggregation and glycosylation.
Optimized lead compound through the stages necessary to allow it to be tested in animal and human trials.
Involvement of pharmacokinetic studies, toxicology testing, and formulation development.

Identifying Bioactive Molecules

Biologically active small molecules have often been discovered by testing a single compound at a time, but such an approach is obviously highly inefficient and cost intensive in terms of both reagents and personnel time. As a result, HTS technologies have been developed to screen large numbers of compounds simultaneously, typically through the miniaturization and automation of assay protocols. What constitutes high throughput will vary depending on technical considerations and the screener’s economic situation, but generally, the number of compounds involved may range from tens of thousands to several million. High-throughput screening assays can be divided into two main classes: “pure protein” and “cell-based”. Pure protein screens generally have optical assay readouts that monitor enzymatic or binding activity. For instance, fluorescence polarization or FRET techniques are commonly used to screen for compounds that affect binding between protein partners. In pure protein assays, every compound screened should have equal access to the target. However, the membrane permeability characteristics of any active molecules that are identified may subsequently pose a major concern if the target is intracellular.

Target Identification

When biologically active small molecules are identified through chemical screens, particularly forward genetic screens, a substantial amount of work is often required to identify the molecular target. Ignorance of the target does not preclude clinical or research use of the molecule; indeed, many clinical agents have been used effectively even when their targets were not known (e.g. aspirin, nitroglycerin, fumagillin, epoxomycin).

Hypothesis-driven Target Identification

For well-characterized cellular pathways, it is sometimes possible to deduce the target of a small molecule by comparing data from across the field. For instance, characteristic phenotypes induced by the small molecule may permit the assignment of the target to a previously identified complementation group. Subsequent hypothesis-driven testing of potential targets can then be undertaken.

With affinity labeling, molecules may be radioactively or chemically labeled; they may be synthetically modified with reactive groups to promote covalent attachment to the target or tagged with specific moieties to facilitate detection. Standard protein fractionation and detection techniques can then be used to identify proteins from crude extracts that are specifically labeled by the molecule. Examples of drugs whose cellular targets were determined by such methods include acetylcholine, the anti-angiogenic agent fumagillin and the antifungal lipopetide echinocandin . Affinity purification of putative targets from cellular extracts is accomplished with molecules immobilized on a solid support used either as a column matrix or as a bead slurry. Small molecules for which cellular targets were identified through affinity purification include the immunosuppressant FK506, the kinase inhibitor purvalanol and the anti-inflammatory compound SB 203580.

Additional studies involving affinity chromatography were instrumental in further characterizing the biochemistry of FK506 and purifying the target of the structurally related compound rapamycin. A natural technological development of affinity-based target identification involves probing proteome chips with small molecules.

Genomic Methods of Target Identification

A. Yeast three-hybrid system

The yeast two-hybrid system has been widely used to study protein–protein interactions and the approach was adapted to create a yeast three-hybrid system for identifying protein–ligand interactions. The method relies on the use of a hybrid “bait” ligand consisting of the query molecule linked to a known ligand. A protein fusion between the known ligand’s receptor and a DNA  binding domain serves as the “hook”, while the “fish” is a protein fusion between a transactivation domain and the target protein. If the target protein interacts with the query molecule, the “fish” and “hook” will be brought together by the “bait” and transcription from a reporter gene is activated. Hence it should be possible to identify the target of the query molecule by cloning a library into the “fish” domain and screening or selecting for activity of the reporter.

A three-hybrid approach was recently used to identify targets of various kinase inhibitors. For each inhibitor, a hybrid ligand was synthesized by attachment to methotrexate and the hook protein was a LexA–DHFR fusion. A three-hybrid screen of a human cDNA library with a purvalanol B–methotrexate ligand identified several (but not all) known purvalanol targets and also several new candidate targets, and all identified targets were kinases. Affinity chromatography and enzymatic assays confirmed 12 of 16 novel candidate targets identified in the cDNA screens.

B. Induced haploinsufficiency

One genomic approach to target identification relies on the premise that altering the gene dosage of the target will affect sensitivity to the small molecule. For example, inactivating one copy of the target gene in a diploid organism would in many cases be expected to increase sensitivity. This method of identifying drug targets through induced haploinsufficiency was established by Giaever et al., who constructed and screened a set of 233 heterozygous yeast deletion mutants for those demonstrating increased sensitivity to known drugs. Each mutant was chromosomally tagged with a unique oligonucleotide and the mutants were pooled and grown in the presence of tunicamycin, at a level of drug that is sublethal for wild type. The relative number of each mutant in the pool was monitored at various time points by polymerase chain reaction (PCR) amplification and fluorescence labeling of all tags in the pool, followed by hybridization of the PCR-generated probes to an oligonucleotide microarray. The fluorescence intensity generated at each spot on the array permitted quantitation of the relative abundance of each corresponding heterozygote in the pool. Mutants unaffected by tunicamycin showed no reduction in signal over time, whereas tunicamycin-sensitive heterozygotes showed varying decreases in signal. In this study, one known and two new loci were identified and confirmed as involved in tunicamycin resistance.

C. Expression profiling

Expression profiling was recently employed in identifying the targets of a class of small-molecule antagonists of FK506 in yeast cells subjected to salt stress. These molecules (termed SFKs for suppressors of FK506) were identified in a chemical genetic screen for molecules that rescue yeast growth in the presence of high salt and FK506 [34]. Expression profiling results obtained with SFK2-treated yeast suggested that the Ald6 p pathway was a target of SFK2 and haploinsufficency screening supported this hypothesis. Overexpression of ALD6 was found to suppress the effects of SFK2–SFK4 on growth and the ability of SFKs to inhibit Ald6 p in vitro was subsequently demonstrated. In addition to using DNA microarray technologies in conjunction with haploinsufficiency studies, it is likely that gene expression profiling will be used increasingly as a primary means of identifying the targets of bioactive small molecules. Many research groups have used expression profiling to identify characteristic patterns of gene expression (“fingerprints”) that are associated with certain disease states or biological pathways. If the patterns are sufficiently unique, then it is sometimes possible to assign previously uncharacterized mutants to specific cellular pathways or complementation groups based on their expression profiles. Analogously, by profiling cells grown with and without a small-molecule modulator, it may be possible to identify genes or pathways that are affected by the small molecule.

LEAD OPTIMIZATION

In analog design, molecular modifications of the lead compound can involve one or more of the following strategies:

  1. Identification of the active part (pharmacophore)
  2. Functional group optimization
  3. Structure activity relationship studies
  4. Bioisosteric replacement
  5. Design rigid analogs
  6. Homologation of alkyl chains or alteration of chain branching, design of aromatic ring position isomers, alteration of ring size, and substitution of an aromatic ring for a saturated one or the converse.
  7. Alteration of stereochemistry, or design of geometric isomers or stereoisomers.
  8. Design of fragments of the lead molecule that contain the pharmacophoric group (bond disconnection).
  9. Alteration of interatomic distances within the pharmacophoric group or in other parts of the molecule.

IDENTIFICATION OF THE ACTIVE PART (PHARMACOPHORE)

Any drug molecule consists of both, essential and nonessential parts. Essential part is important in governing pharmacodynamics (drug receptor interactions) property while non-essential part influences pharmacokinetic features. The relevant groups on a molecule that interact with a receptor are known as bioactive functional groups. They are responsible for the activity. The schematic representation of nature of such bioactive functional groups along with their interatomic distances is known as pharmacophore.

Once such pharmacophore is identified, structural modification can be done to improve pharmacokinetic properties of the drug. For example, the presence of a phenyl ring, asymmetric carbon, ethylene bridge and tertiary nitrogen are found to be minimum structural requirement for a narcotic analgesic to become active. Similarly the presence of two anionic sites and one cationic site must be present in cholinergic agent.

FUNCTIONAL GROUP OPTIMIZATION

The activity of a drug can be correlated to its structure in terms of the contribution of its functional group, to the lipophilicity, electronic and steric features of the drug skeleton. Hence by selecting proper functional group, one can govern the drug distribution pattern and can avoid the occurrence of side-effects.

STRUCTURE ACTIVITY RELATIONSHIPS

The physiological action of a molecule is a function of its chemical constitution. This observation is the basis of interpretation of activity in terms of the structural features of a drug molecule. Generalized conclusion is then can be made after examining a sufficient number of drug analogs.

BIOISOTERIC REPLACEMENT

The concept of bioisosterism is derived from Langmuir’s observation that certain physical properties of chemically different substances are strikingly similar. These similarities were rationalized on the basis that carbon monoxide and nitrogen both have 14 orbital electrons and similarly, diazomethane and ketone both have 22 orbital electrons.

Bioisosteres are substituents or groups that have chemical or physical similarities and which produce broadly similar biological properties. Bioisosteres have been classified as either classical or nonclassical. Classical bioisosteres are those which have similar steric and electronic features and have the same number of atoms as the substituent moiety for which they are used as replacement. Non classical bioisosteres do not obey the strict steric and electronic definition of classical isosteres and they don not have the same number of atoms as the substituent moiety for which they are used as a replacement. These isosteres are capable of maintaining similar biological activity be mimicking the spatial arrangement, electronic properties, or some other physicochemical property of the molecule of functional group that is critical for the retention of the biological activity.

DESIGN OF RIGID ANALOGS

Imposition of some degree of molecular rigidity on a flexible organic molecule (e.g., by incorporation of elements of the flexible molecule into a rigid ring system or by introduction of a carbon-carbon double or triple bond) may result in potent, biologically active agents that show a higher degree of specificity of pharmacological effect. The three-dimensional geometry of the pharmacophore can be determined and the key functional groups are held in one steric disposition, or in the case of a semirigid structure, the key functional groups are constrained to a limited range of steric dispositions and interatomic distances. By the rigid analog strategy, it is possible to approximate “frozen” conformations of a flexible lead molecule that, if an enhanced pharmacological effect results, may assists in defining and understanding structure activity parameters.

HOMOLOGATTION OF ALKYL CHAIN OR ALTERATION OF CHAIN BRANCHING, CHANGES IN RING SIZE, AND RING POSITION ISOMERS.

Change in size or branching of an alkyl chain on a bioactive molecule may have profound and sometimes unpredictable effects on physical and pharmacological properties. Alteration of the size and shape of an alkyl substituent can affect the conformational preference of a flexible molecule and may alter the spatial relationships of the components of the pharmacophore, which may be reflected in the ability of the molecule to achieve complementarily with its receptor or with the catalytic surface of a metabolizing enzyme. The alkyl group itself may represent a binding site with the receptor through hydrophobic interactions, and alteration of the chain may alter its binding capacity. Position isomers of substituents even alkyl groups on an aromatic ring may possess different pharmacological properties. In addition to their ability to affect electron distribution over an aromatic ring system, position isomers may differ in their complementarily to receptors, and the position of a substituent on a ring may influence the spatial occupancy of the ring system.

ALTERATION OF STEREOCHEMISTRY AND DESIGN OF STERIOISOMERS AND GEOMETRIC ISOMERS.

In the case of chiral molecules, one enantiomer would be expected to demonstrate pharmacological activity and the other enantiomer should be expected to be pharmacologically inert, is not valid.

At high doses (R)-enantiomer selectively stimulated presynaptic dopaminergic receptor sites, while at lower doses, it selectively stimulated postsynaptic receptor sites. In contrast, the (S)-enantiomer stimulated presynaptic dopamine receptors and at the same dose level, it blocked postsynaptic dopamine receptors. Thus, this enantiomer exhibits a bifunctional mode of dopaminergic attenuation: that of presynaptic agonism and postsynaptic antagonism.

FRAGMENT OF THE LEAD MOLECULE

Design of fragments of a lead molecule is based on the premise that some lead molecules, especially polycyclic natural products, may be much more structurally complex than is necessary for optimal pharmacologic effect. A bond disconnection strategy may be employed, in which bonds in the polycyclic structure are broken or removed or destroy one or more of the rings.

VARIATION IN INTERATOMIC DISTANCES.

Alteration of distances between portions of the pharmacophore of a molecule or even between other portions of the molecule may [produce profound qualitative and quantitative changes in pharmacological actions.

LEAD DEVELOPMENT

The screening cascade is designed to decrease the number of compounds examined at each level in order to ensure that compounds with flaws are removed as early as possible. The cascade, also referred to as a screening tree, begins with in vitro profiling and then transitions into in vivo studies designed to determine a compound’s pharmacokinetic profile and demonstrate efficacy in an appropriate animal model.

At the top of the cascade, compounds are screened for activity against the biological target and a threshold of interest is generally set to determine if compounds are active enough to warrant further investigation. Potency is, of course, an important issue, as dosing requirements are lower for compounds that are more potent. All other things being equal, compounds with higher potency can be dosed at lower levels, decreasing the likelihood of side effects. A compound with target potency of 5nM in theory could be provided to a patient at a significantly lower dose than a compound serving the same function but with a potency of 5μM.

Once a compound has satisfied the potency criteria, selectivity and physicochemical properties criteria are typically examined. Nature has developed exquisite systems to accomplish very specific tasks with highly selective systems, but many of these systems overlap structurally, and this can have a significant impact on the biological properties of a given test compound. Thus, the next biological screening step in a typical screening cascade is often an assessment of a compound’s potency at biological systems that are closely related to the target of interest. The Kv1.5 channel, for example, is a voltage-gated potassium channel that has been the target of research programs atrial arrhythmia, and many compounds have been identified that can block this channel with a high level of potency.82 There are, however, over 70 other voltage-gated potassium channels with varying degrees of similarity to the Kv1.5 channel, and undesired activity at any of these related channels could create unwanted side effects in human or animal studies. For example, the Kv1.5 channel is closely related to the hERG channel. Blockade of the hERG channel has been linked to torsade de pointes and sudden cardiac death,83 so any compound moving forward in this area would need to be counterscreened for hERG activity in order to ensure that advancing compounds do not present a risk of sudden cardiac death in a clinical setting. This is a rather extreme example of the importance of proper selectivity, but it should be clear that failure to achieve proper target selectivity in this area represents a significant barrier to moving a program forward.

Similarly, there are over 500 known kinase enzymes,84 and any drug discovery program designed to target a single kinase, or even a family of kinases, has an associated risk of identifying compounds that are active at multiple members of this large family of related enzymes. In order to mitigate this risk, kinase programs routinely screen test compounds against panels of related kinases in order to understand the risks associated with off-target activity.

In general, compounds that are potent at the target of interest, but are also potent at a variety of other targets (“promiscuous compounds”), do not move forward in a drug discovery program, as the risk of undesired (or unpredicted) side effects is too high. The level of selectivity required, however, is dependent on the program, the nature of the potential side effect presented by off-target activity (off-target activity leading to excessive hair growth might be tolerable, whereas sudden cardiac death through poor hERG selectivity is not), the target patient population, duration of treatment (some side effects only appear upon extended exposure to a drug), and a variety of other factors. Overall, target selectivity is a major factor to consider.

An active and suitably selective compound, however, is not necessarily a good drug candidate. Physicochemical properties also play a major role in determining whether or not a compound is suitable for further investigation. In vitro screens designed to predict absorption, distribution, metabolism, and excretion (in vitro ADME) are generally performed early in a program in order to ensure that candidates reaching the drug development pipeline are “druglike” in nature. Compounds that have poor aqueous solubility, for instance, are often difficult to develop as drugs. In order for a drug to exert an influence on a biological target, it must be soluble in biological fluid at a level consistent with its potency. Thus, the level of solubility required for a given compound is directly linked to its potency. As potency increases, the requisite solubility decreases, as less drug is required to provide the intended effect. Solubility also has a direct impact on absorption, as a compound must be soluble in biological fluids in order for it to successfully pass through a biological membrane and reach its intended target.

The ability of a compound to penetrate cellular membrane (its permeability) can also be a determining factor in the success or failure of a given candidate compound. If a compound is potent, selective, and soluble, but unable to pass through a biological membrane, it may not be able to reach the target of interest and fail to demonstrate the desired efficacy. Orally active drugs must be absorbed in the gastrointestinal tract, and drugs that target intracellular system must also pass through the cell membrane in order to reach their intended targets. Extracellular targets, of course, do not face this added issue, but CNS drug candidates face the added complexity of required permeability through the blood–brain barrier (BBB). Additionally, there are efflux pumps (e.g., P-glycoproteins (Pgps))85 designed to remove xenobiotic material that can limit permeability, preventing efficacy. The inability of a compound to penetrate a cell membrane represents a significant issue that could prevent further investigation of the candidate compound.

Metabolic and chemical stability are also important considerations. If all of the previously mentioned criteria are met, and a compound is able to enter the body, but is immediately metabolized, efficacy studies will fail to show the desired results. However, the relative rate of metabolism that can be allowed for a successful compound depends on the goals of the project. If, for example, the goal is to develop a new antibacterial agent, then high-metabolic stability will likely be desired so that the potential drug candidate will be available in the circulation long enough to kill the invading organism. If, however, the goal is to develop a new surgical anesthetic, metabolic stability may be less of an issue, as it may be desirable for drug efficacy to fade rapidly upon termination of dosing regimens.

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Pre-Clinical Trial

Pre-Clinical Trial
Process Development
Engineering Run
Upstream
Downstream
Toxicological Study
Analytical
Production Support
Stability Study

 


Upstream

It includes operations related to cell expansion steps. Under cell expansion a single vial of frozen cell is cultured exponentially into larger systems until a large scale terminal reactor is reached.

List of Equipments/facilities/reagents:

  1. Cell processing center
  2. Containment barrier isolator (CBI)
  3. Isoclean Healthcare Platform Isolator (HPI-G3)
  4. CradlePro-Iso
  5. Laboratory Scale Tide Motion Bioreactor: Cel Cradle
  6. Laboratory Scale Fermenter: StirCradle
  7. Pilot and Production Scale Tide Motion Bioreactor - TideCell
  8. Pilot/Production Scale Fermenter: StirCradle-Pro
  9. BioNOC II carriers
  10. Cell Culture Media and Supplement
  11. Cell Culture Equipment (Biological Safety Cabinets, Versati centrifuge, laboratory shaker

Cell Line Development and Clone Selection

In the course of cell line development, a company-internal selection of host cells, expression vectors, transfection and selection methods takes place. The selection of the expression system is determined by its ability to ensure a high productivity and defined quality criteria. The expression system most commonly used for the production of monoclonal antibodies or recombinant proteins are Chinese Hamster Ovary (CHO) cells. The first proteins produced by CHO-derived cell lines were recombinant interferons and tissue-type plasminogen activator (tPA). In 2010, approximately 70% of all recombinant proteins have been produced in CHO cells. High productivity and posttranslational processing are the criteria for cell line selection after cell transfection. Other factors, such as growth behavior, stable production, cultivation in serum-free suspension media, adaptive behavior, amplification, clone selection and possible risk assessment are taken into account as well.  Li et al. (2010) and Costa et al. provide good overviews of current methods for optimizing expression vectors and transfection methods. Several methods for cell line optimization are employed prior to cell clone selection to improve and ensure product quality. One important parameter of product quality is the reproducibility of the glycosylation profiles. These depend on the respective cell clone, medium and cultivation conditions. Commonly used methods are RNAi and gene deletion technologies. Other approaches in cell engineering help to avoid ammonium and lactate accumulation and improve cell growth. Codon optimization and various approaches for gene-amplification via different selection markers serve to optimize the cell line. Further approaches include metabolic engineering or anti-apoptosis enhancements. Costa et al. (2010) provide additional resources on cell engineering and its aspects.

The subsequent selection of the best suited cell clone is one of the most important steps of the upstream development process as variations in the production cell line during clinical development constitute a major process change. Such a change requires an additional proof of product comparability. The cell clones considered for final production have to fulfil the required product quality, processability and volumetric productivity. Criteria for selection are: growth, cell-specific and volumetric productivity, glycosylation profiles, development of charge variants, aggregate formation, protein sequence heterogeneity and clone stability among others. In addition, metabolic characteristics can be exploited for cell clone selection, as well as their stability, robustness, high viability and low lactate or ammonium generation. At the end, process performance in the bioreactor decides which clone will be used for production and which will be saved as a backup. Running this selection process in the shortest time possible presents a big challenge. Especially product quality, productivity, and the metabolic profiles of the cells strongly depend on cell culture conditions.

Transient gene expression (TGE) as another possibility of protein production should be mentioned at least briefly. TGE is used to produce recombinant proteins over a short period of time following a DNA transfer into single-cell suspension cultures. There is no need for genetic selection of transfected cells and therefore a lot of time is saved. Today, TGE is applied as a screening-tool for drug development since only small doses of potential drugs are needed within a short amount of time . IgG yields of 2–80 mg/L have routinely been achieved with this process [56,57] which can be increased of 250–300 mg/L . For industrial purposes, transient gene expression is promising regarding its high time-savings in cell line development. For manufacturing processes, it is likely to be tried with low-dose proteins before an application for antibody-derived products will be attempted.

Media Development and Optimization

Media optimization processes have led to progress in commercially available media over the last decades. Early cell culture media like Ham’s F10 or Dulbecco’s Modified Eagle Media were based on blood serum supplements. These include a complex mixture of unknown components. In the 1970s and 1980s, many serum-free media were developed in order to provide better defined cultivation media like IMDM or CMRL medium. Further improvements eliminated all animal-derived components to avoid pathogen contaminations and thus resulting in chemically defined media.

Media development is a key factor in improving productivity and growth behavior of cells but it also influences product quality . Today, commercially available media present the basis for media development towards optimized conditions for a process using a specific cell line. The optimal blend of media components has to be solved individually due to the high diversity of cell lines, processes, media components, interactions of components and metabolic pathways. Effective media development depends strongly on the choice of optimization tools. The most common strategies are based on: component titration, media blending, spent media analysis and automated screening.

A combination of these methodologies provides the most rational way of media development. The development process itself includes a screening process to identify important components, followed by an optimization step and a verification of the process . Standardization of these development approaches led to a platform-based media development.

Traditionally, media were developed by changing one factor at a time. In order to reduce experimental efforts, DoE and high-throughput methods are applied in industrial development processes. As an example, a top down approach is described by Ma et al. (2009). Media development needs to be optimized for each cell line individually but its establishment as a platform process in development leads only to an improvement of USP. There is still some potential for optimization which can be drawn upon, if necessary.

Approaches for feed media development include variations in the concentration of the basal medium, nutrient consumption, accumulation of impurities and a balance of cell growth and volumetric productivity . Generally, methods for feed media optimization are the same as in basal media optimization, including the use of DoE.


Downstream

It is also called as the purification process. This step is mainly focus on the capture and isolation of the target molecule and the removal of the impurities. The ultimate goal of down processing is to manufacture drug products for a safe and effective delivery.

List of equipment/facilities/reagents:

  1. Airflow Containment (Pharmacon Downflow Booths, Ceiling Laminar Airflow Units, Laminar Floor Horizontal Trolley/ Laminar Flow Vertical Trolley, Enterprise Laminar Flow straddle Units, ESCO Garment storage cabinet, Ventilated Balance Enclosure)
  2. Isolation Containment (Aseptic Containment Isolator, Containment Barrier Isolator, General Processing Platform Isolator, Isoclean Healthcare Platform Isolator, Streamline Compounding Isolator, Weighing and Dispensing Containment Isolator.
  3. Cross Contamination Facility Integrated Barrier (Biopass Pass Through, Infinity Air Shower Pass Box, Cleanroom Air showers, Infinity Cleanroom Transfer Hatch, Infinity Pass Boxes, Soft Capsule Soft Wall Cleanroom, Dynamic Passboxes and Dynamic Floor Label hatches.
  4. Barrier Isolation System (CradlePro-Iso, Cytotoxic Safety cabinets)

Chromatographic Separations

After cell harvesting by centrifugation or filtration, a chromatographic separation unit is used to isolate antibodies from fermentation broth. Protein A chromatography is one of the most important unit operations for antibody capturing. It distinguishes itself by high selectivity towards IgG-type antibodies, high flow rate and capacity. The dynamic binding capacity ranges from 15–100 g mAb/L resin depending on antibody, flow rate and adsorbent. The degree of purity is consistently higher than 95% . Process-related impurities like HCP, DNA, media components and virus particles are removed . One of the major advances in recent years of process development consists in a better integration of chromatography to the overall manufacturing process. Elution conditions of the initial Protein A capture step are adjusted to the following unit operation in order to enter a subsequent virus inactivation step or an ion exchange chromatography. This eliminates any need for buffer exchange between these unit operations and it is one example for a successful integration of single separation operations during the last decades.

Problems exist in form of Protein A leaching and non-specific binding of impurities like HCP and DNA. Leached Protein A reduces the binding capacity of Protein A chromatography and needs to be removed in subsequent purification steps.The amount of bound impurities depends on the adsorbent, composition of cell culture harvest, column loading and washing conditions. Tarrant et al.  and Shukla et al.  published studies on HCP interacting with different Protein A matrices and the product.

Cation exchange chromatography (CEX) presents an alternative to Protein A chromatography. It requires a pH shift of the feed and a decrease of the conductivity before loading onto the column in order to optimize the dynamic binding capacity. Older CEX processes distinguished themselves by a capacity of 20–30 g/L which cannot cope with new increasing product titers. Optimizations of the resin resulted in capacities as high as 100 g/L at high flow rates und purity. This technique can be used for antibodies with a basic isoelectric point . Antibody variants, e.g., charge variants or aggregates, can be removed as well as most negatively charged impurities. The costs of CEX are approximately one fifth of the costs for Protein A chromatography.  Synagis and Humira are two examples of commercially available mAbs which are purified by an application of CEX as capture step.

Pharmacy and chemistry theme. Female scientific researcher putting flask with liquid solution in gas chromatography

Non Chromatographic Separations

  • Membrane processes are one of the most important unit operations in biopharmaceutical processing. In USP, microfiltration membranes filtrate media, buffer and gases; in DSP they can be used as initial harvest operation for removal of biomass particles prior to chromatographic operations and DNA from cell cultures Ultrafiltration membranes with a range of 1–100 nm are used to concentrate and diafilter biomolecules . In order to ensure virus clearance, symmetric membranes are necessary with a narrow pore size distribution of 20–50 nm. The separation mechanism is mainly based on molecular weight and to a lesser extent on shape and charge. Other membranes frequently used are depth filters or high performance tangential flow membranes which can be neutral or charge. Kumar et al. (2013) separated biomolecules based on their charge by charged ultrafiltration membranes
  •  The former function of membranes as selective barrier for filtration is extended towards a selective adsorption of molecules to separate them according to their chemical behavior. This relatively new development in membrane technology is called membrane chromatography. These are symmetric microfiltration membranes functionalized with specific ligands attached directly to the convective membrane pores. Diffusive pores are eliminated, mass transfer of biomolecules depends on convection and the binding capacity is largely independent of flow rates. Significant advances have recently been made in developing high permeability and high capacity sterile filters by application of composite membranes. Membrane adsorbers are used for polishing applications to remove contaminants. Viruses, endotoxins DNA, HCP and leached Protein A binds to the membrane at neutral to slightly basic pH and low conductivity. Additionally, salt-tolerant membrane adsorbers have been developed for viral clearance as well as HIC membranes which have comparable dynamic binding capacities to conventional HIC resins. Other development trends investigate the possibility to apply membrane adsorber in capture and purification of large biomolecules and will focus on new designs of structures for bind-and-elute processes. Other fields in need of optimization concern flow distribution, membrane size distribution and thickness.
  • Research activities on aqueous two-phase extraction (ATPE) show potential applications of this process for separation of cells and undissolved components, of impurities and product. ATPE is considered a simple and low-cost technology compared to Protein A chromatography. It has advantages in scalability, can be applied in continuous processes and has a high capacity. Unfortunately, there is still a limited understanding of molecular mechanisms of ATPE. Other downsides are difficulties regarding its use as a platform step due to complex interactions of the multiple components involved. Another problem might be a sensitivity to feed stream variability. In spite of this, strategies for ATPE design and process implementation are being developed and already applied in purification processes of recombinant proteins.
  • Precipitation can also be used for protein purification in industrial scale. Current research indicates possible applications for product concentration and the separation of product and impurities. Volume limitations of the subsequent unit operation can be accommodate. Membrane filtration removes the supernatant and is followed by dissolving the precipitate in a preferred buffer volume. This filtration process can be carried out either by dead-end filtration in lab-scale or by cross-flow filtration in industrial scale . Centrifugation can be used as a substitute for filtration. Another application of precipitation could be as a purification step before a capture by CEX. HCP and media components would be separated before a chromatographic capturing would take place. Antibodies can be separated by either ammonium sulfate precipitation or co-precipitation with negatively charged polymers. Another possible application consists in a co-precipitation of several impurities with positively charged polymers. Those impurities include acidic HCP, DNA, and residual media components.
  • Crystallization is mostly applied in protein structure analysis and is already used as a cost effective and scalable purification procedure for small molecules. Examples are the purification of low molecular weight substances like amino acids or industrial enzymes, like industrial lipase, or ovalbumin. In insulin purification, crystallization is applied as polishing step benefitting formulation aspects of higher stability. Its application in antibody purification is limited due to their size and heterogeneity. A possible establishment in the purification process could be  performed inexpensively and with large volumes. Crystallization would benefit formulation due to a higher stability of the final product in crystalline form. It might be a suitable alternative to chromatography or ultra- and diafiltration . However, this procedure is currently not ready for use. Only three recent studies presented potential µl-scale crystallization of whole antibodies [110] and Smejkal et al.  reported a successful crystallization of an IgG1-type antibody in a stirred L-scale.

Analytical

This process is an overlapped of the downstream and the upstream processes and is responsible for ensuring all raw materials, procedures and areas are released for production. It also performs testings on raw materials, in-process and final product validation with environmental monitoring product activities.

List of Equipments/facilities/reagents:

  1. PCR Thermal Cyclers (Conventional Thermal Cyclers, Real-time PCR systems)
  2. PCR Sample Handling (Microplate shakers, PCR cabinets)

Production Support

This comprises a variety of support for technical tasks such as filling or finishing operations.

List of Equipments/facilities/reagents:

  1. ASEPTiCell
  2. Vial Washing
  3. Automatic Trayloaders
  4. Depyrogenation Tunnel
  5. Filling/Stopper Inserting Machines
  6. Capping Machines
  7. Automatic Loading/Unloading Equipment
  8. External Vial Washer

Stability Testing

Stability testing is critical during preclinical and clinical testing to establish an accurate assessment of the product being evaluated. This is also done to test the extent of the drugs’ biological activity.

List of Equipments/facilities/reagents:

  1. Sample Storage and protection solution (Lab Refrigerators and Freezers, Voyager – Remote monitoring programming, datalogging software, ESCO PROtect Wireless Monitoring System)
  2. Laboratory Thermostatic Products (Irefrigerated incubator, Laboratory Ovens)
  3. SubliMate Freeze Dryer or lyophilizers
  4. Stability Isolators

Changes in biopharmaceutical stability due to degradation, chemical or physical instability can alter protein folding and the protein 3-dimensional structure, affecting efficacy. A host of complex analytical methodologies may be required to determine stability and these must be justified and validated. Biological activity assays and the quantitative detection of degradation products are also required to comply with Good Manufacturing Practice (cGMP) compliant stability program. Environmental factors (such as temperature, exposure to oxygen or changes in pH), adsorption onto surfaces and interactions with excipients can all affect stability. In order to demonstrate the stability of biologics, forced degradation studies should be designed and conducted to determine stability-indicating methods suitable for ongoing stability studies.

Biopharmaceutical stability studies for biologic therapeutics are essential to assess sensitivity to factors that could cause aggregation and degradation which impacts biologic activity, product safety, and quality.

Biopharmaceutical Stability Testing Methods:
  • Amino-acid analysis
  • Aggregation analysis
  • Gel Electrophoresis (1-D and 2-D SDS-PAGE reducing and non-reducing)
  • Western blot
  • Isoelectric focusing
  • Capillary electrophoresis (CE)
  • Peptide fingerprinting by Chromatography and Mass Spectrometry (MS)
  • Peptide mapping and sequencing by LC-MS/MS including S-S (Di-sulphide) bridge mapping analysis
  • Total Protein Quantification
  • Carbohydrate and Glycosylation Studies
  • Post-translational modifications (PTM)
  • Liquid chromatographic patterns
  • Immunochemistry techniques
  • cGMP Cell-based Bioassays
  • Spectroscopic profile
  • Higher Order Structure Characterisation by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), Infra -Red Spectroscopy (FTIR)

Toxicological Testing

This step is performed by a) studying the accidental exposure to a substance, b) in vitro studies using cells or cell lines and c) in vivo exposure on experimental animals .

List of Equipments/facilities/reagents:

  1. PCR Thermal Cyclers (Conventional Thermal Cyclers, Real-time PCR systems)
  2. PCR Sample Handling (Microplate shakers, PCR cabinets)
  3. VIVA Universal Animal Containment Workstations
  4. VIVA Dual Access Animal Containment Work Stations
  5. VIVA Bedding Disposal Animal Containment Work Stations
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Clinical Trial Approval

Clinical Trial Approval

Investigational New Drug (IND) is the general application to administer a new drug to humans. Studies in human can only start upon the approval of IND by the Food and Drug Administration (FDA) and an institutional review board (IRB).

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Clinical Trials

Clinical Trials
Phase I
Phase II
Phase III
The main goal is to assess the safety of the candidate drug in humans. It is usually conducted in a small number of healthy volunteer (100 or less). Pharmacokinetic, pharmacodynamics, and safe dosing range are evaluated. This phase is necessary to determine if the candidate drug will move on to the next phase.
The effectiveness of the drug candidate is evaluated in this phase using 100-500 patient volunteers with the condition under study. Optimal dose strength, short term side effects and risk are also analyzed. This phase is necessary to determine if the candidate drug will move on to a larger phase III trial.
Demonstrate safety and efficacy in a large group of patient usually 1000-5000 patients or more across numerous clinical trial sites around the world. Planning for the full scale production of medicine is conducted on this stage. Drugs at this stage must be produced in a CGMP facility and is the final phase in which the process can be changed before commercial production.

Phase I

During Phase 1 studies, researchers test a new drug in normal volunteers (healthy people). In most cases, 20 to 80 healthy volunteers or people with the disease/condition participate in Phase 1. However, if a new drug is intended for use in cancer patients, researchers conduct Phase 1 studies in patients with that type of cancer.

Phase 1 studies are closely monitored and gather information about how a drug interacts with the human body. Researchers adjust dosing schemes based on animal data to find out how much of a drug the body can tolerate and what its acute side effects are.

As a Phase 1 trial continues, researchers answer research questions related to how it works in the body, the side effects associated with increased dosage, and early information about how effective it is to determine how best to administer the drug to limit risks and maximize possible benefits. This is important to the design of Phase 2 studies.


Phase II

In Phase 2 studies, researchers administer the drug to a group of patients with the disease or condition for which the drug is being developed. Typically involving a few hundred patients, these studies aren't large enough to show whether the drug will be beneficial.

Instead, Phase 2 studies provide researchers with additional safety data. Researchers use these data to refine research questions, develop research methods, and design new Phase 3 research protocols.


Phase III

Researchers design Phase 3 studies to demonstrate whether or not a product offers a treatment benefit to a specific population. Sometimes known as pivotal studies, these studies involve 300 to 3,000 participants.

Phase 3 studies provide most of the safety data. In previous studies, it is possible that less common side effects might have gone undetected. Because these studies are larger and longer in duration, the results are more likely to show long-term or rare side effects.

Drugs at this stage must be produced in a CGMP facility and is the final phase in which the process can be changed before commercial production.

For biosimilars or biologics a change in bioprocess method such as changing bioreactors from roller bottles to TideCell may require a bridging study or redoing phase 3

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Marketing Approval

Marketing Approval

The New Drug Application (NDA)/ Biologics License Application (BLA) is submitted to FDA for marketing approval. These application contains results and data analysis from the previous clinical trials.

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Post-Marketing Surveillance

Post-Marketing Surveillance

Production plants of active pharmaceutical ingredients, and drug products are primarily batch operated multi purpose manufacturing plants. At this facilities, commercial supplies of API intermediates, APIs and drug products are manufactured before being packaged, labeled, and distributed.

List of equipment/facilities/reagents:
  1. Airflow Containment (Pharmacon Downflow Booths, Ceiling Laminar Airflow Units, Laminar Floor Horizontal Trolley/ Laminar Flow Vertical Trolley, Enterprise Laminar Flow straddle Units, ESCO Garment storage cabinet, Ventilated Balance Enclosure)
  2. Isolation Containment (Aseptic Containment Isolator, Containment Barrier Isolator, General Processing Platform Isolator, Isoclean Healthcare Platform Isolator, Streamline Compounding Isolator, Weighing and Dispensing Containment Isolator.
  3. Cross Contamination Facility Integrated Barrier (Biopass Pass Through, Infinity Air Shower Pass Box, Cleanroom Air showers, Infinity Cleanroom Transfer Hatch, Infinity Pass Boxes, Soft Capsule Soft Wall Cleanroom, Dynamic Passboxes and Dynamic Floor Label hatches.
  4. Barrier Isolation System (CradlePro-Iso, Cytotoxic Safety cabinets)
  1. ASEPTiCell
  2. Vial Washing
  3. Automatic Trayloaders
  4. Depyrogenation Tunnel
  5. Filling/Stopper Inserting Machines
  6. Capping Machines
  7. Automatic Loading/Unloading Equipment
  8. External Vial Washer

Primary production is the processing of raw materials to create active pharmaceutical ingredients (APIs) and ancillary substances used in pharmaceutical formulations. The final API in the pharmaceutical that produces the therapeutic effect, should meet pharmacopoeial requirements. Primary manufacturing may involve either chemical or biological processes requiring different types of production facilities, technologies, skills, and knowledge. The manufacture of active ingredients is the most expensive aspect of pharmaceutical production because of the necessary investment in capital equipment, process development, and quality assurance systems.

Pharmaceutical Ingredients: These are portions of any drug considered active with which the reactions and results vary (depending on the drug’s administered dosage). Most drugs are made of more than one kind of pharmaceutical ingredients.

Components:

API or Active Pharmaceutical Ingredients -- the main ingredient known to have a biologic/ therapeutic activity.

Excipient or ancillary substance – the inert or inactive substance combined with a drug.

Secondary production is the large-scale processing of finished dosage forms (like tablets, capsules, parenteral, syrups etc.) from raw materials or intermediate products, from both local and imported sources. Production of sterile preparations (examples are as follow: injections, antibiotics, and intravenous fluids) and non-sterile preparations (oral solids, liquids, and topical drugs) can be carried out with either locally produced or imported packing materials. Just like primary production, this stage must be accomplished to clear-cut specifications. It necessitates modern, high-speed, precision equipment to produce drugs in their final form, in large quantities and at very low unit costs, meeting international GMP standards.

Tertiary production includes packaging and labelling finished products from primary and secondary sources into bulk packs, smaller dispensing packets, bottles, or course-of-therapy units for individual use. The initial quality of the pharmaceutical product established in the earlier phases of production must be maintained in the tertiary and final step, so ensuring high quality standards through rigorous operational procedures is important. This type of production can be developed first in many countries as a positive contribution that also builds industrial skill and experience. Tertiary production also addresses specific local needs for certain formulations, labeling, and packaging.