Product Design and Description of the Probiotic

Product design for probiotics starts with the selection of a strain of bacteria or yeast that has characteristics thought to be associated with healthy functioning of human body systems. Selected strains usually have been isolated from healthy individuals or animals. Although the route of administration is usually oral, intended for the gastrointestinal system, probiotics are also being studied for vaginal use, and administration by application to the skin has also been suggested [1]. Selected strains should be characterized using scientifically valid techniques.

Strain identification, source, and history. Probiotics intended as biological drugs must be accurately identified by genus and species. The particular strain identification should be included, with references to publications, technical reports, and other sources. It is important that strain designation conform to previously published identifications, to facilitate the tracing of relationships to precursor or related strains. If a known strain is altered in its genetic or phenotypic characteristics during its development as a live bio-therapeutic, it should be given a new strain name, and traceability to the parent strain should be documented. In the case of combination products, each strain and its source should be characterized separately and their selection should be controlled by acceptance criteria before combination in a final product.

The complete strain history, from its isolation to the establishment of a master strain, should be reconstructed and documented as accurately as possible. Reconstruction of the history may be difficult when strains have been passaged many times, reisolated, exchanged between laboratories, or stored in ways that could result in the “drifting” of important characteristics. Properties of the cell substrate and events during research and development can affect the resultant product quality and safety [2]. Diligence is required in determining if and when the strain has been stored or manipulated under conditions that may have exposed it to adventitious infectious agents, such as the use of mammalian components (e.g., serum, hydrolysates, and enzymes) or simply preparation in an environment where other microorganisms are present without safeguards to prevent or detect inadvertent contamination.

A selected strain's potential for pathogenicity should be assessed. One indication of a strain's potential for pathogenicity is the historical epidemiology. Conventional wisdom is that probiotic strains are avirulent by virtue of their presence in normal gut or mucosal flora. However, there have been reports of systemic infection in persons with underlying disease or who underwent separate invasive procedures [3, 4]. During development, a risk assessment should be conducted that considers the potential for inadvertent disease due to excess dose exposure, accidental systemic exposure, and/or administration to vulnerable hosts. Appropriate animal studies should be performed to determine the virulence with respect to the route of administration and the number of viable cells in the dose.

Testing of strain identity. There are many in vitro microbiological, biochemical, and immunological techniques [1, 3] available that may be used in appropriate combination to identify and characterize the selected strains, as follows.

1. Bacteria and yeast are typically identified to the species level by their fermentation patterns and products. Any end product associated with the indication should be identified in the master strain.

2. Inherent resistance to gastric acidity or bile-salt activity may be critical to the effectiveness of a strain intended for oral administration.

3. The antibiotic-susceptibility profile is an important and useful characterization of strain identity.

4. Antimicrobial activity against pathogens or competition for adhesion may be important.

5. Resistance to spermicides should be studied for vaginal indications.

6. Interactions with primary cultures of epithelial cells should be studied.

7. Induction of cytokines by epithelial cells, PBMCs, and spleen cells should be tested.

Definitive identification will rely on genetic techniques, which may be the only way to differentiate strains within the same species. Genome sequencing identifies definitively to the species level. DNA hybridization can be used to identify genus, species, and possibly strain if the type of strain or a reference strain is designated. 16S ribosomal RNA sequencing has proved useful in identifying differences between strains, as well as in identifying the species [3]. Strains of the same species often can be differentiated by the fingerprints that result from PCR amplification of isolated DNA and by comparison of the ladders that result from electrophoresis and staining. The presence of plasmids should be determined, and, if they are present, it should be determined whether the plasmid encodes antibiotic-resistance genes.

Cell banks. After selection of the strain, the initial step in manufacturing is to establish a master seed-cell substrate to be preserved as a master cell bank. The identified strain is first prepared as a pure culture or clone. Selected strains may require adaptation to modified culture conditions that enable large-scale production. Once the strain is confirmed to be a pure culture with the desired characteristics, it is preserved in a qualified cell bank.

A cell bank provides a common source for production, to ensure consistency in the manufacturing process and in the resulting product [2]. Cell banks for production are usually set up in a 2-tiered system consisting of a master cell bank derived from the initial clone and a working cell bank derived from the master cell bank. Cell banks should be as large as possible (in terms of the number of units) to avoid remaking the master cell bank or replicating the working cell bank too frequently. The acceptance criteria for the working cell bank should be defined prospectively, and the same criteria should be applied to subsequent production of the working cell bank.

The preparation of the cell bank should be documented in laboratory notebooks or in a formal document system. Preservation is commonly done by freezing at ultralow temperatures or by freeze-drying. The effect of freeze-drying on cell characteristics, such as viability, or on the production of the desired end products should be studied.

The establishment of the cell bank should be traceable both backward to the original source and forward to each production lot. This is accomplished by identifying the cell banks by part numbers and lot numbers. There should be a system for inventory control. Removal of units for purposes other than production, such as for stability studies or research, should be documented. Individual units should be indelibly labeled.

The loss of a master cell bank would be a serious setback in product development. The ability to produce the product without interruption should be ensured by taking measures to protect the critical strain or clone from accidental loss due to fire, power outages, or catastrophic weather. The GMP process indicates restricted access to the master cell bank and the working cell bank for only supervisory, quality-control, and authorized production personnel. Usually, this means lock-and-key security or equivalent access control and possibly alarm systems. The master cell bank should be stored in >1 secure location.

Manufacture of the Live Biotherapeutic Product

In the United States, a strain intended to be developed and marketed as a therapeutic product or for prevention of a specific disease condition is defined as a live biotherapeutic product and will be regulated as a biological drug. The regulation of biological products by the US Food and Drug Administration (FDA) primarily uses the Biologics License Application process. The Biologics License Application is used for all products regulated under the Public Health Service Act [5].

Manufacturing process. A flow chart and text description should show the steps taken in culture expansion, propagation, harvest, and purification and should depict all critical inputs and outputs that are used to control the process. Examples of process inputs that define the manufacture of products containing microorganisms include propagation settings (time, temperature, oxygen tension, and agitation rate) and controls during harvest (speed and time for centrifugation, time and flow rate during filtration, and flow rate or exchange rate during tangential flow). Research will indicate which parameters are most critical to ensuring a consistent process and product quality. Product stability may depend on control of buffer composition and storage temperatures.

Process development usually proceeds in parallel with preclinical and clinical studies, and the results of clinical studies can confirm the reliability of the process as designed. The final process design should be in place by the time of the pivotal clinical studies. Process changes made late in clinical development may require additional studies to ensure that the product quality has not been adversely affected.

Process-output measurements ensure that the process is performing as expected and will result in a consistent product. Typical measurements include growth monitored by optical density, pH change, and colony-forming units per unit of volume. Purity of the culture is a critical parameter and should be assessed by staining and subculture of the inoculum and of the final harvest. The yield of the harvest can be expressed as mass or viable cell count per unit of volume (or both). Criteria for determining when to harvest, such as a fixed time or target optical density, should be described.

Harvest and purification might be combined in a single step or a sequence of steps. Yields typically decrease with successive purification steps. Once the desired purity is achieved, stabilizers may be added to the final suspension.

Formulation. The active ingredient of a live biotherapeutic is the harvested and concentrated live organism. This is formulated into a dosage form that is convenient for use, ensures an accurate dosage content, and ensures the quality of the active ingredient. Usually, a concentrated harvest is adjusted by dilution, or a dried preparation is blended with other dry ingredients, such as stabilizers or fillers. Liquid preparations to be frozen may be stabilized with cryoprotectants. Uniformity of the dosage form is ensured by monitoring of weights and volume during the filling of vials, capsules, or sachets or during compression into tablets. Tablets and capsules may be enteric coated to ensure passage through the stomach and dissolution in the intestine. Powdered product may be supplied with prepackaged buffered reconstituting solutions.

Control of quality and consistency. Because major process changes often result in changes in the product quality, the process should be designed to require minimal adjustments. When deviations from the design occur, these are documented for evaluation during product-release decisions. Master production and control records (MCPRs) are a powerful tool for ensuring against deviations. The MCPR describes each step in manufacture, the required inputs, and the expected ranges for outputs. The MCPR also controls the manufacturing environment by specifying locations and equipment. Product integrity and quality is ensured by defining the conditions and times for in-process steps in which the product may be held and by specifying the storage conditions.

A lot- or batch-specific executed MCPR is called a “batch record.” The batch record describes all component materials in the final product and materials used during manufacture that are intended to be removed during purification. Materials for production should be used only if they are compliant with the manufacturer's own specifications. The batch record contains all test results for in-process and final testing or provides a direct, traceable link to records of testing. The batch record notes individual pieces of equipment used in manufacture and allows traceability to records of use, maintenance, and cleaning. Finally, the batch record describes the processing locations and provides dates that allow traceability to records of environmental conditions during processing.

A particular component of biological drug products deserves additional comment [6]. Potable tap water is not acceptable for use as a component of pharmaceutical products, regardless of the intended indication or route of administration. Purified water should be used for sterilized propagation media and in formulation. Sterile purified water should be used for formulation of nonparenteral dosage forms.

Control of contamination and cross-contamination. The manufacture of biological products uses principles of aseptic processing because the component materials and final product may be degraded by contaminating microorganisms. Biologics are usually not heat sterilized, because active ingredients are heat labile. The entire process must be “aseptic,” or bio-burden must be controlled.

Water is controlled for bioburden or sterility, as appropriate to the stage of manufacture and the indicated use of the final product. Air is controlled for bioburden and nonviable particulates. Component materials may require sterilization before use and should also be monitored for bioburden. Product-contact equipment should be sterilized, or sterile disposable equipment can be used. All areas and surfaces in the manufacturing environment where open steps are performed should be monitored for bioburden.

A related concern is prevention of cross-contamination with other products manufactured in the same areas. Cleaning and disinfection reduce bioburden and ensure removal of previous products. Multiple products should never be manufactured in the same areas at the same times.

Specifications for Live Biotherapeutic Products

Specifications are acceptance criteria to which active ingredients and drug product must conform before they can be released as products for human use. Acceptance criteria are dependent on the methods used to obtain the results. Specifications should be established for critical materials used in the manufacture of human drugs and biologics, for intermediates, and for the final products. Specifications for product release are chosen to confirm the product quality, rather than to characterize the product.

Control of the harvested live biotherapeutic. The harvested live organism is the active pharmaceutical ingredient. Before formulation, the active pharmaceutical ingredient is tested and released against acceptance criteria that ensure the quality of the final product.

Specific identity testing is performed to confirm the strain and its critical characteristics. For recombinant strains, products of transgene expression should be verified.

Microbiological purity should be assessed for the purified active pharmaceutical ingredient and after high-risk manufacturing steps that might allow extraneous contamination. Testing methods, such as the United States Pharmacopoeia microbial-limits tests [7], may be used to ensure freedom from extraneous organisms. Purity testing at the stage of the active pharmaceutical ingredient is more sensitive than is testing after dilution to prepare the final dosage form. Test methods are selected to detect the most likely contaminants. Although sterility of the live product and intermediates is not claimed, extraneous bioburden should be controlled by GMP and should be monitored at significant points in the manufacturing process.

Finally, an accurate quantity of viable organisms is determined using sample dilution and colony counts. Yield of the active ingredient should be measured as colony-forming units per product unit. This value guides formulation and is a useful monitor of process consistency.

Control of dosage form. The regulations for biologics [8] list the product qualities that must be evaluated for each lot of product: potency, general safety, sterility, purity, and identity. For some qualities, the methodology is also specified.

Potency is the specific ability or capacity of the product to effect a given result. Potency is defined [9] as a quantitative measure of an attribute linked to relevant biological properties. The simplest measure of potency for a live biotherapeutic is colony-forming units per dose unit. The accuracy of the measure of colony-forming units will be determined by the sample size, homogeneity of the sample, and number of replicate plates per diluted sample. If an end product of the microbe is associated with or predictive of therapeutic activity, then an assay of the end product is also appropriate. A semiquantitative assay of an expressed product may be sufficient when combined with measurement of colony-forming units or another quantitative biochemical assay. Potency is often measured against a reference material or a specially prepared batch of active pharmaceutical ingredient.

The general safety test method specifies doses given by intraperitoneal injection to mice and guinea pigs observed for adverse effects for 7 days. The general safety test is intended to detect extraneous toxic contaminants in the final product. It is not intended as a test of inherent product toxicity and must be modified for products that are toxic by nature (e.g., live organisms given parenterally in high doses). A regulatory exemption from the general safety test may be requested, provided that GMP and quality controls are in place.

Sterility testing is usually not appropriate for live biotherapeutics. A modification of the test method might be designed with consideration of the special characteristics of the organism, such as generation time or anaerobic growth requirements. Sterility testing may be waived for products that are administered orally [10].

Purity testing is repeated for the dosage form, to rule out extraneous contamination during formulation and finishing steps. For certain dosage forms, dissolution or resistance to gastric acid are appropriate tests to ensure that the active ingredient will reach its most effective site for activity.

For final products that are dried or freeze-dried, the moisture content is a critical parameter that can affect the viable cell count and product quality during its shelf life. Moisture content is measured for any product prepared in a dried form. Identity testing should differentiate the product from similar products produced in the same manufacturing areas.

Stability. Stability studies are performed to ensure that the product remains safe and effective for its intended use throughout its specified shelf life. The dosage form should be tested in the actual container or closure system intended for marketing and should be stored at the recommended temperature.

The FDA will review data that support the recommended usage and administration instructions in the package insert as follows.

1. If a powdered drug product is supplied with a liquid for reconstitution, the stability of the reconstituted product should be studied.

2. If a powdered or liquid product is combined with a consumer-supplied matrix, support for the recommendation must be presented as stability data. Limits on the kinds of diluents or appropriate foods should be stated in the package insert.

3. Consumers should be instructed on the limits of storage time after reconstitution or combination with any recommended diluent or matrix.

A stability protocol defines the kinds of testing to be performed and a schedule for each test. Most release testing is included, but every test may not be required at every time point. The kinds of assays to include are those that test potency, purity, moisture, and bioburden.

Summary

Expectations for product development of a live biotherapeutic product do not differ from those for other FDA-regulated products that consist of live microorganisms, such as live vaccines or microbial vectors for gene therapy or for tumor vaccines. Recommendations for steps in early product development include the following.

1. Select and characterize a strain as a master seed and establish a master cell bank.

2. Carefully document the production lot(s) for nonclinical studies and establish working specifications.

3. Repeat production with few or no modifications to the product clinical lots.

4. Confirm or modify specifications on the basis of clinical results.

5. Begin stability studies early.

State-of-the-art technology should be used to characterize the selected master strain and the master cell bank. Each lot of a working cell bank should meet the selected acceptance criteria.

Detailed records of the production of nonclinical batches should be used to create an MCPR for production of clinical materials. The development of specifications is an iterative process intended to improve production and control.

Final specifications for the active pharmaceutical ingredient and the final dosage form will be established on the basis of clinical results and manufacturing performance. Stability results may also drive final specifications and will determine the critical labeling elements for the marketed product.

Product development proceeds in parallel with clinical development. Changes are expected during development as knowledge is gained. Careful documentation of each phase of development is critical to support a successful product approval.