PARENTERALS

What are Parenterals? Are sterile, pyrogen free preparations injected through skin or mucous membrane into internal body compartments. Sterile products: Are dosage forms of therapeutic agents that are free of viable microorganisms. Parenterals Opthalmics Irrigating preparations

Parenteral administration of drugs involves the injection of therapeutic agents, in the form of solutions, suspensions or emulsions, into the body. In doing so, one of the major barriers to drug entry (the skin) is breeched. Parenteral formulations have been officially recognized since the mid 19th century when morphine solution appeared in the 1874 addendum to the British Pharmacopoeia (1867).

Routes of parenteral administration There are several different routes by which parenteral products may be administered There are three routes by which parenterals are most frequently administered: Intravenous (IV) Intramuscular (IM); and Subcutaneous(SC)

Intravenous route Involves administration of the parenteral formulation into a vein, usually a large proximal vein. The veins are located beneath the subcutaneous tissue, embedded within the muscle. Achieves a rapid and predictable response Ensures 100% drug bioavailability

Both large- (up to 500 ml) and small- (up to 10 ml) volume formulations may be administered intravenously. Formulations are usually solutions or emulsions (in which the size of the disperse phase is small, less than 1 um).

Care must be taken regarding the rate of administration of the parenteral formulation. If the administration is performed too quickly, an excessive concentration of drug at the target organ may result, leading to drug-induced shock. Training is required to ensure that the dosage form is actually administered to the vein and that puncture of the vein is avoided.

Intramuscular route This involves administration into a muscle, usually the Gluteal (buttocks), Vastus lateralis (lateral thigh) or Deltoid (upper arm) muscles.

The volume of injection is small, usually 13 ml or up to 10 ml in divided doses. Faulty injection technique may lead to local muscle damage. IM injection results in relatively rapid absorption, second only to IV with respect to the time taken for the onset of action. Drug absorption from aqueous solutions is greater than from aqueous suspension or non-aqueous (oil-based) solutions of drugs. IM injections are usually used for controlled-release formulations.

Subcutaneous route This involves administration into the subcutaneous tissue, a layer of fat located below the dermis. There is a slower onset of action and sometimes less total absorption of therapeutic agents when compared to the IV or IM routes of administration. The volume of injection is typically circa 1 ml Hypodermoclysis: large-volume parenteral solutions (electrolyte or dextrose, up to 1000 ml) may be infused subcutaneously. This technique is only employed when there is difficulty in accessing a vein.

Viscous formulations are not generally administered subcutaneously SC administration is the route of choice for the administration of insulin.

Miscellaneous Routes These include: intradermal (ID) intra-arterial (IA) intrathecal (IT) intradural and extradural intracardiac (IC) routes.

Intradermal route This is the injection into the dermal layer of the skin. The ID route is generally used for diagnostic purposes, e.g. for the diagnosis of allergy and for the tuberculin test. Absorption is slow and limited Only small volumes may be injected, circa 0.1 ml.

Intra-arterial route The parenteral formulation is injected into an accessible artery. This route requires specialist training to administer therapeutic agents as if the artery is missed, possible damage to adjacent nerves may result. The IA route is used to administer radiopaque media to visualize organs, e.g. heart, kidney. It is used to administer anticancer drugs to ensure that the highest possible concentration of drug reaches the target organ.

Intrathecal (IT) route This route is used to administer therapeutic agents to the cerebrospinal fluid to ensure that the appropriate concentration of drug is obtained at this site (e.g. for the treatment of infection).

Intradural and extradural routes Intradural and extradural administration is employed to achieve spinal anaesthesia.

Intradural administration involves injection of the therapeutic agent within the dural membrane surrounding the spinal cord. Extradural administration involves injection of the therapeutic agent outside the dural membrane and within the spinal caudal canals.

Intracardiac (IC) route The intracardiac route involves injection of the formulation directly into the muscles of the heart. This route is normally used whenever there is a cardiac emergency.

Advantages An immediate physiological response may be achieved (usually by the IV route). This is important in acute medical situations, e.g. cardiac arrest, anaphylactic shock, asthma. Parenteral formulations are essential for drugs that offer poor bioavailability or those that are rapidly degraded within the gastrointestinal tract (e.g. insulin and other peptides).

They offer a method to administer drugs to patients who are unconscious or uncooperative or for patients with nausea and vomiting (and additionally dysphagia). As trained medical staff primarily administer parenteral formulations, there is control of both the dosage and frequency of administration. Local effects may be achieved using parenteral formulations, e.g. local anaesthesia. Parenteral formulations provide a means by which serious imbalances in electrolytes may be corrected (using infusion solutions). Parenteral formulations may be readily formulated to offer a wide range of drug release profiles, including: rapidly acting formulations (generally drug solutions that are administered IV) long-acting formulations (generally drug suspensions, or solutions in which the drug is precipitated out of solution at the injection site, administered by the IM or SC routes).

In patients who cannot consume food, total parenteral nutrition offers a means by which nutrition may be provided using specially formulated solutions that are infused into the patient.

Disadvantages The manufacturing process is more complicated than for other formulations due to the requirement for aseptic technique. The level of training of staff involved in the manufacture of parenteral formulations is high specialist equipment is required to ensure that the finished product specification is achieved.

Skill of administration is required to ensure that the dosage form is administered by the correct route. If a parenteral suspension, which is designed for administration by the IM or SC route, is incorrectly administered by the IV route, a pulmonary micro-capillary blockage may occur leading to a blockage in the flow of blood at that site.

Parenteral formulations are associated with pain on administration. If the patient is allergic to the formulation (the therapeutic agents and/or the excipients), parenteral administration will result in both rapid and intense allergic reactions.

It is difficult to reverse the effects of drugs that have been administered parenterally, even immediately after administration. This is not strictly the case with other routes of administration, e.g. oral, transdermal.

Formulation considerations for parenteral formulations Parenteral formulations may be categorised as Solutions (aqueous or oil-based), Suspensions (aqueous or oil-based) or Emulsions.

Solubility of the therapeutic agent With respect to the formulation of pharmaceutical products, therapeutic agents may be categorised into three groups: Good solubility, in which the therapeutic agent is freelysoluble in the chosen solvent (either aqueous or oil-based) at the concentration required in the parenteral product. In this case a parenteral solution is a possible formulation option. Moderate solubility but insufficient to produce a solution in conventional solvents (e.g. water, oil). In this scenario, the use of co-solvents may sufficiently increase the solubility of the therapeutic agent in the vehicle to produce a parenteral solution containing the required drug concentration. This is generally the preferred strategy for therapeutic agents of moderate solubility.

However, if required, the therapeutic agent may be formulated as a suspension, although one cautionary note regarding this approach is the potential recrystallisation of soluble drug during storage, a phenomenon that may affect the physical stability of the preparation. Low solubility in the chosen vehicle: this property gives a challenge to formulator. The steps which may be taken require selection of alternative solvent which gives high solubility.

Preferred route of administration If there is a preferred route of administration for the parenteral product, this will directly influence the nature of the parenteral product. IV products must be aqueous solutions and, furthermore, must not precipitate in the blood stream following administration. Emulsions may also be administered by this route provided the particle size of the internal phase is sufficiently small.

Parenteral suspensions (aqueous or oil-based) and oil-based parenteral solutions must be administered either subcutaneously or intramuscularly. Aqueous solutions may also be administered intramuscularly or subcutaneously. There are other restrictions for those less commonly used routes of parenteral administration that are specific for each route.

Volume of dose to be administered The volume of product to be parenterally administered will directly affect the type of formulated product. Large-volume parenterals (up to 500 ml) are administered intravenously (although the SC route of administration is infrequently used for this purpose). Small-volume parenterals may be administered by all routes (bearing in mind the restrictions of oil-based and suspension formulations).

Onset/duration of action Formulations administered intravenously will have an immediate pharmacological effect. The rates of drug absorption from the other main routes of administration (SC and IM) are slower. The absorption of therapeutic agents from aqueous solutions when administered by the IM or SC routes is faster than from oil-based solutions, oil-based suspensions and aqueous suspensions. When injected subcutaneously, the onset of action of soluble insulin (aqueous solution) is rapid (circa 30 minutes), peaks between 2 and 4 hours and has a duration of action of up to 8 hours. Intermediate/long-acting insulins (aqueous suspensions), when administered subcutaneously, have an onset of action of 12 hours, a peak action between 4 and 12 hours and a duration between 16 and 35 hours.

Physicochemical properties of the therapeutic agent The physicochemical properties are important determinants of the stability and absorption of the therapeutic agents when formulated as parenteral suspensions (aqueous or oil-based). Solid-state properties Solubilities of insoluble salt forms Particle size

All parenteral formulations may be formulated using an aqueous vehicle, an oil vehicle or a hydroalcoholic vehicle,

The choice being determined by The required solubility of the active agent in the formulation and the Desired type of formulation.

Vehicles For Injection AQEOUS VEHICLE: Used more Frequently, isotonic (to blood) to which drug may be added at the time of use. Used primarily to effect solubility of drugs and / or reduce hydrolysis. Ethyl alcohol; polyethylene glycol(liquid) and propylene glycol

Non Aqueous vehicles: employed for the production of: non-aqueous parenteral solutions of therapeutic agents that are water-insoluble non-aqueous parenteral suspensions of therapeutic agents thatare water-soluble and/or exhibit aqueous instability the internal phase of parenteral emulsions.

Fixed oils are predominantly used as non-aqueous vehicles (e.g. corn oil, cottonseed oil, peanut oil, sesame oil) non-aqueous esters may be used, e.g. ethyl ethanoate: Sesame oil is generally the oil of choice as it is more stable. Oils must be free from rancidity and must not contain mineral oils or solid paraffins. Two major problems associated with the use of non-aqueous pharmaceutical solutions are: Pain/irritation on injection.

It should be noted that the viscosity of fixed oils increases at lower storage temperatures. affect the ease of administration by injection and the pain/irritation at the site of injection. Patients may exhibit sensitivity to the oils and therefore the oil used in the formulation must be explicitly stated on the label/patient information.

Co solvents: co-solvents are employed whenever the solubility of the drug in water (or occasionally in oil-based vehicles) alone is insufficient for the required application. Examples of co-solvents used in parenteral Formulations include: glycerol ethanol (high concentrations of ethanol are known to produce pain on injection) propylene glycol polyethylene glycol 400.

The concentration of co-solvent used should be sufficient to render the drug soluble within the formulation (over the shelf-life of the product) but should not be irritant or toxic to the patient.

Types Of Water For Injection Water for Injection Highly purified Water used as a vehicle for injectable preparations which will be subsequently sterilized. Can be stored for less than 24 hr at RT or for longer times (5 or 80 C). Need to meet USP sterility test since used in products which will be sterilized. Need to meet USP Pyrogen test. Maximum 1 mg/100 ml Total solids. May not contain an added substance

Sterile Water for Injection USP (SWFI) Appropriate type of water used for making parenteral solutions prepared under aseptic conditions and not terminally sterilized.

Needs to meet USP Sterility Test. Can contain an added Bacteriostatic agent when in containers of 30 ml or less. Single dose containers no exceeding 1000ml.

Bacteriostatic Water for Injection USP SWFI containing one or more suitable Bacteriostatic Agents. Multiple dose containers not exceeding 30 ml Not the vehicle of choice when need larger than 5ml due to toxicity of Bacteriostatic agent.

Sterile water for irrigation: Wash wounds, surgical incisions, or body tissues.

Sterile water for inhalation

Parenteral Added Substances Antibacterial agents: Required to prevent microorganism growth. Limited concentration of agents. Phenylmercuric Nitrate and Thimersol 0.01%. Benzethonium chloride and benzalkonium chloride 0.01% Phenol or cresol 0.5% Chlorobutanol 0.5% Effectiveness varies with formulation. Binding of parahydroxybenzoic acid with macromolecules

FORMULATION OF LVPS

Large volume parenterals can be categorised into: 1)Hyperalimentation solutions 3)Peritoneal dialysis solutions 2)Cardioplegia solutions 4)Irrigation solutions

Hyperalimentation solutions: Administration of large amounts of nutrients (carbohydrats, amino acids)to a patient who is unable to take food orally for several weeks. Cardioplegia solutions: Used in heart surgery to help prevent ischemic injury to the myocardium.These are typically electrolyte solutions where the electrolyte composition is intended to maintain diastolic arrest. Peritoneal dialysis solutions: Continuous infusion into abdominal cavity and withdrawal to aid remove toxic substances from the body(drug or chemical toxicity) or to accelerate the excretion function normal to the kidney(in case of kidney dysfunction).These solutions contain glucose and have an ionic content similar to extracellular fluid. Irrigating solutions: To irrigate, flush, and aid in cleansing body cavities and wounds.

Classes of Parenteral additives: Antimicrobials: Generally added to products that are not terminally sterilised. Trend to eliminate preservative wherever possible. If used,interaction between product componenet and the preservative should be considered.(Proteins biniding with thiomersal). Preservative efficacy testing sohould be carried out. Eg:Thiomerosal(0.01),Benzlykonium chloride(0.01), Metacresol(0.1-0.3) Antioxidants: Ascorbic acid(0.01-0.03), Sodium bisulphite(0.1-1), Sodium metabisulphite(0.11).

Buffers: Parenteral product should be sufficiently buffered to maintain the desired pH to maintain product stability. Buffers commonly used in paarenterals include acetates,phosphates and citrates.Amino acids are used as buffers for polypeptide injections. Chelating agents: Are added to complex and inactivate metals such as copper, iron, zinc etc. coming into the products through sources like water, containers and closures etc. Eg: EDTA derivatives , citric acid etc Tonicity adjusting agents:

Nonisotonic solutions if given in amounts greater than 100ml, can cause hemolysis or crenation of RBCs. NaCl, Dextrose are commonly used to adjust the isotonicity.

Vehicles Aqueous vehicles: WFI is most widely used solvent for parenteral preparations. It should be prepared and stored in a mannar to ensure purity and freedom from pyrogens. Inorganic compounds are removed from water mainly by reverse osmosis, distillation, deionisation or combination of these. Apart from SWFI, Nacl(0.9%), Dextrose(5%), and variety of electrolyte and nutrient solutions can also be used for reconstitution. Nonaqueous and mixed vehicles: Major class is fixed oils. Commonly used oils are cottonseed oil, corn oil, peanut oil etc. If any of these oils is used in the formulation, it should be stated on the label. Parenteral Nutrition Components are in elemental or pre-digested form Protein as amino acids CHO as dextrose Fat as lipid emulsion Electrolytes, vitamins and minerals

Delivery of nutrients intravenously, e.g. via the bloodstream. Central Parenteral Nutrition: often called Total Parenteral Nutrition (TPN); delivered into a central vein Peripheral Parenteral Nutrition (PPN): delivered into a smaller or peripheral vein

Parenteral Nutrition Macronutrients & Micronutrients

Parenteral Base Solutions Carbohydrate

Available in concentrations from 5% to 70% D30, D50 and D70 used for manual mixing

Amino acids Available in 3, 3.5, 5, 7, 8.5, 10, 15, 20% solutions 8.5% and 10% generally used for manual mixing

Fat 10% emulsions = 1.1 kcal/ml 20% emulsions = 2 kcal/ml 30% emulsions = 3 kcal/ml (used only in mixing TNA, not for direct venous delivery)

Considerations: Type of feeding formula and tube Method (bolus, drip, pump) Rate and water flush Intake energy and protein Tolerance, complications, and corrective actions Patient education

FORMULATION & MANUFACTURING OF SVP

Small volume parenteral (SVP) : USP defines it as an injection that is packed in containers labeled as containing 100 ml or less. USP categories it as follows:1) Solution or emulsion of medicaments suitable for injection 2) Dry solid or liquid concentrates containing no additives which , upon the addition of suitable solvent , yield solution for injection 3) Preparation same as 2 but containing one or more additional substances

4) Suspensions of solids in a suitable medium which are not to be injected intravenously or into the spinal column 5) Dry solid which, upon the addition of suitable vehicles, become sterile suspensions

Formulation Principles: Route of administration Since parenteral perparation are introduced directly into the intra- or extracellular fluid compartments, the lymphatic system, or the blood, the nature of the product & the desired pharmacological action are factors determining the particular route of administration to be employed. Appropriate volume :IV - >10ml Intraspinally <=10 ml IM up to 3ml SC up to 2ml Intradermal up to 0.2 ml Selection of the solvent :The choice of the solvent or vehicle is directly related to the intended route of administration of the product . IV & intraspinal injection dilute aqueous solution I.M and subcutaneous :-oily solution, co solvent solution, suspension and emulsion, it should be hypertonic solution Intraspinal injection must be isotonic. Water for injection, USP :- is the solvent of choice for making parentrals.it must be prepared fresh by distillation or by reverse osmosis and contain no added substance. when it is not possible to use fully aqueous solution for physical or chemical reason, the addition of solubilizing agents or co solvent(glycerin, ethanol, propylene glycol or polyethylene glycol) may be necessary. Commonly used additives in parenteral products Antimicrobial agents:- benzalkonium chloride, benzyl alcohol, chlorobutanol, chlorocresol, phenol, phenylmercuric nitrate and acetate, thiomerosal.

Antioxidants:- acetone sodium bisulfite, ascorbic acid, BHA, BHT, cysteine, tocopherols, sodiumbisulfite, meta bisulfite, glutathione. Chelating agent:- EDTA, Japan does not allow addition of EDTA in parenteral product. Buffers:- acetic acid and salt, pH 3.5-5.7 citric acid and salt, pH 2.5-6.0 glutamic acid, pH 8.2-10.2 phosphoric acid and salt, pH 6.0-8.2

Tonicity adjustment:- dextrose, Nacl, sodium sulphate Surfactant:- polyoxyethylene sorbitan monooleate, sorbitan monooleate, lecithin , polysorbate 80. Buffers: Although buffer assure the stability of pH of solution , the buffer system itself can alter other properties such as kinetic & solubility aspects. Buffer can act as general base catalysts & cause degradation of the drug substance. Such a mechanism occurs with a number of amine & amine derivatives in systems containing polycarboxylic acids ( e. g. citric , tartaric & succinic). In such case , the degradation of vitamin B1 increases with increase in citrate buffer concentration. Antioxidants: Agents that have a lower oxidation potential than the drug , & thus can be preferentially oxidized , are called antioxidant .

They functions in at least two ways (1) by being preferentially oxidized & thereby gradually consumed or (2) by blocking an oxidative chain reaction in which they are not usually consumed. Antimicrobials:- they are often added to unit dose solutions which are not sterilized at the terminal stage of their manufacture.

They fall into 5 basic classes of chemicals: the quaternary ammonium compounds, alcohols, esters, mercurials, & acids. The majority of formulation falls in 3 categories

1) Solution 2) Suspension/ dispersion 3) Solids for constitution

A) Preparation of small volume parenteral solution-

It involves dissolution of all the ingredients in an appropriate solvent system. ( water, cosolvents, nonaqueous systems such as vegetable oils) It is convenient for end use as iv, im, sc Content uniformity is easily obtained during manufacture & administration Clean room condition must be maintained to minimize particulate & microbial contamination In addition to soluble drug , a sterile solution may contain following agents-

1) Osmotic pressure adjusters NaCl, mannitol 2) Bacteriostatic agents- benzyl alcohol 3) Buffering agents- phosphates, acetates, citrate. 4) pH adjusters- NaOH, HCL 5) Antioxidants bisulfite, ascorbate, citrare 6) Chelating agents- EDTA

Operation 1- nonsterile formulation

1. Place WFI into clean, vented , glass or stainless steel pressure tank. 10% extra to cover losses due to evaporation, seal the pressure tank 2. Heat WFI to 121 c for 20 min, then cool to 60 c 3. Remove & place 30% of WFI separately & save for final volume adustment . 4. To the remaining WFI , add & dissolve sodium phosphate dibasic ( anhydrous). 5. Cool the solution to R.T, add & dissolve water soluble drug & preservative. Check pH, if required adjust to 6.8 to 7.0 with 1N NaOH. 6. Make up the final volume & mix well. Operation 2- sterilization 1) Sterilize by filteration through a sterile sterilizing membrane, 2) Collect it via sterile tubing into a sterile, clean , closed, vented, stainless steel tank or glass vessel. Operation 3- sterile subdivision Aseptically subdivide & apply closure to container & seal. The preferred method of sterilization is terminal heat or radiation sterilization in the package. However , many organic medicinals lack stability in solution during irradiation or heating at high pressure steam autoclaves. An altenative method has been to steam or dry heat sterilize those portion of the formula that are not heat labile . In such case , additional thermal sterilization of the vehicle is done by autoclaving both the vessels from operation 1 , step 3 & 4, for 30 min at 121c and remaining procedure is followed same from step 5 as described earlier . Some pharmaceutical companies export concentrated solution (5x) of final product to subsidiary foreign companies. this procedure satisfies some regulatory requirement & provide an economical advantage for shipping bulk rather than packaged solution. The offshore company then makes arrangement for dilution & subdivision. Small volume parenteral formulation Rx Dosage 50 mg 9 mg category Active drug Preservative

Hydrocortisone sodium phosphate Benzyl alcohol

Sodium citrate Sodium bisulfite Sodium hydroxide WFI qs.

10 mg 3 mg 1 mg Ad 1.0 ml

Buffering agent Antioxidant pH adjustment solvent

B) Preparation of suspension It is more complicate in composition , difficult to process & sterilize than solution. Usually portion of the suspension composition is prepared & sterilized separately. And then combined aseptically to yield a final sterile bulk product for aseptic subdivision. The suspended product, which has particle size of 5 to 10m or larger will be filtered out by 0.2 or 0.45m filter. The solid active ingredient may be sterilized prior to compounding into a suspension. Drug with low m.p & heat sensitive are not stable to DHS & autoclaving is not the common practice since aq. solubility is increased with temp & there is chances of solid to dissolve , decompose & recrystallize on cooling in diffferent crystal forms. Ethylene oxide treatment is another way but residuals of the toxic gas & its by-products may remain on the powder Fitzpatrick mill are commonly used under stringent clean, sterile condition , to reduce the particle size of a sterile solid. Air attrition mills such as the Jetomizer, micronizer, Jet pulverizer are commonly employed,. The air used must be sterile .Milling is done in a laminar flow area or clean room .

Once the sterile solid of proper particle size is available, the manufacture of a sterile suspension can commence. Operation 1- Preparation of saturated sodium chloride & Drug paste 1) Place all NaCl into taired , clean , callibrated , Pyrex glass wide mouth bottle & water in a quantity sufficient to dissolve all the NaCl. 2) Mix well by magnetic bar or electrical mixer. 3) Slowly add required amount of insoluble drug 4) Wet the solid by mixing 5) Seal the bottle by a stopper containing a s.s type 316

6) Hold the sealed container for autoclaving

Operation 2 Na CMC solution 1) place predetermined quantity of WFI to Dissolve the Na CMC 2) Start agitation with Lightnin mixer to create a vortex. 3) Slowly, add Na CMC into vortex 4) Mix vigorously until gelatnous lumps are visible, while still hot, clarify through a coarse clarification filter. 5) Make up the desired volume of NaCMC with WFI 6) Collect it in clean pyrex bottleplace in a metal can, partially insert a stopper, cover the bottle neck & stopper with kraft paper. 7) Hold the wrapped container for autoclaving. Operation 3- Polysorbate 80 solution 1) Place polysorbate 80 into suitable container. Add WFI to make up the volume 2) partially insert a stopper, cover the bottle neck & stopper with kraft paper. & autoclave Operation 4- Sterile WFI Place into a suitable pyrex glass bottle an excess of WFI , partially insert a stopper covered with parchment paper, place the bottle in a metal can and autoclaving . Operation 5- Preservative solution Dissolve the preservative in WFI , mix well, transfer to a S.S vessel. The following preparations are autoclaved & then placed in a sterile room & cool to R.T 1) Insoluble drug slurry in saturated NaCl 2) NaCMC solution 3) Polysorbate 80 solution it is gently agitated before cooling to R.T to prevent solidification 4) WFI Aseptic formulation in the laminar Flow area-

Aseptically, under LAF , mix sterile polysorbate 80 solution & sterile insoluble drug / saline paste to form a smooth paste, Trasfer sterile Na CMC to above mixer,Aseptically fillter through 0.22 sterilizing membrane, Make up the vol with WFI Aseptic Homogenization

Aseptically homogenize the sterile suspension using APV Gaulin or Bran lubbe mill, recirculate for 5- 10 min without no pressure, then apply pressuer 1500 -2000 psig for 5-10 min, aseptically divert the flow of suspention from the homogenizer to clean, sterile, pyrex glass bottle equipped with sterile teflon stirring bar or sealable mixer. mechanical colloidal mill, such as rotor-startor mill & Koruma mill, ultrsonic mill, combination of ultrasonic mill with mechanical size reduction have become popular.

Aseptic subdivision

Subdivide the suspention into suitable packages, insert a stopper. Sample periodicallly for sterility testing , fill vol, & uniformity of suspention Or obvious extraneous particulate matter. Rx Insoluble drug Polysorbate 80, USP Sodium chloride , USP Sodium carboxymethyl Cellulose Benzyl alcohol WFI 9.00 mg a.q ad 1.0 ml Preservative vehicle Formulation for a sterile Injectable Suspension Dosage 8.00 mg 0.20 mg 6.67 mg 5.00mg category Active drug Surface active agent Tonicity additive viscosity builder

C) preparation of a Freeze dried powder formula Freeze drying is a drying process applicable to the manufacture of pharmaceuticals, biologicals, serums & hormones which are thermolabile or otherwise unstable at aqueous solution but stable at dry state. By removing solvent by the physical process of sublimation , heat sensitive drugs or biologicals can de dried with minimal degradation of the product.

A product to be freeze dried is prepared , steriled & subdivided as a sterile solution or sterile suspension. After the desired amount of material is filled into a container , it is subjected to freezing by following processes. 1) Freezing in standard freezer chests at -18 to -50 c for a prolonged period of time 2) Freezing on refrigerated shelves of a lyophilization chamber at -50 c or below. 3) Freezing in a liquid nitrogen tunnel at a predetermine rate of movement via a moving belt through the tunnel the frozen mass in the lyophilization chember is subjected to vacuum & freeze drying cycle is commenced. The product should be cooled to a temp below its eutectic point The freeze drying process consists of two distinct cycles:

1) Primary drying , which constitutes the bulk of the sublimation process, & removes most of the solvent & forms a cake 2) secondary drying , which removes most of the solvent. The latter part of the cycle usually requires a small amount of external heat input & results in drying the cake thoroughly. Operation 1- Lyophilization chamber preparation 1) If residual material is present , rinse the chamber with hot D.W & wipe down with a lint free cloth 2) Arrange the precalibrated lyophilizer thermocouples uniformly throughout the chamber & condenser. These cold spots are to be monitored at temp control point during steam sterilization. 3) The chamber door is secured & all the external vent valves to the vacuum pumps & vacuum release lines are closed. 4) The thermocouple temp & chamber pressure monitor devices on the chamber are turned on. 5)When all the thermocouples reach 121 c , begin the sterilization timing & it is maintained for a period of time to reach F0 value adequate to sterility. 6) The clean steam line valve & the vent valve are turned off. Chamber chill down The chamber shelves & condenser plates are chilled to below the freezing point of the eutectic of the product to be lyophilized.

Operation 2- Nonsterile solution formulation 1) Place 10% excess WFI into clean, vented, glass lined or S.S pressure tank , heat to 121 c for 20 min , then cool to 30 c 2) Remove 30% WFI , seal the vessel & save for final volume adjustment 3) To the 70% WFI at 30 c , add & dissolve mannitol 4)Add & dissolve water soluble drug & preservative. Check the pH of the solution . Adjust the pH with 6.8 to 7.0 with 1N NaOH or 1N HCL . Make up the final bulk volume Operation 3- solution filteration Sterilize the bulk solution by filtering through sterile sterilizing membrane, with an appropriate nonshedding preclarification filter. Operation 4- Aseptic solution subdivision into sterile container Operation 5- Lyophilization Product Freezing (via the Refrigerated shelf of the lyophilization chamber) 1) Load the product to the product to be lyophilized into the chamber. Stopper the vials partially 2) Place a thermocouple in the center of the solution 3) when all the thermocouples reach the desired temp to crystallization, the condenser plate temp controller is set at -60 c Primary Drying

when all the condenser plate thermocouples reach -50 c , the vacuum pump are turn on . When a pressure reaches 100, allow the product to equilibrate for 1hour , maintaine the shelf temp set point for the duration of the predetermined cycle. Secondary Drying

Increase the shelf temperature to 30 c or a temp that will not degrade the product being lyophilized. Maintain the chamber in this mode for predetermined period of time that guarantees that a minimum level of solvent remains Product stoppering

Within the chamber, the vials can be stoppered under vacuum or pressure of an inert gas such as nitrogen or argon .When the vials being stoppered, the operator shuts off the hydraulic cylinder .this allows the stoppered vials to drop down on the shelf for removal & capping. Formulation for a Lyophillized sterile injectable product

Rx Soluble drug Mannitol NaOH or HCL WFI

Dosage 10 mg q.s q.s pH 6.8 -7.0 1.0 ml

Category Active drug Bulking agent/tonicity agent pH adjustment Solvent

D) preparation of sterile dry fill powder for constitutionMany antibiotic products are marketed as sterile dry filled powders. When a lyophillization process is unacceptable , the alternative process consists of aseptically placing a sterile powder into a sterile vial. This process is known as sterile dry powder filling . It is difficult to achive and maintain powder uniformity when a small quantity of one substance is added to a very large bulk quantity of another substance. This difficulty is compounded by difference in the powder density, particle size distribution, shape of particle, packing , powder cohesiveness, charge . The Turbla is a unique mixer which allows the processor to maintain all of the powder in a single sterile receptacle or mixing container which sealed to exclude air , moisture, and external contamination . The unique blending pattern of the mixer provides excellent uniformity of the blend.

Operation 1- subdivision 1) The sterile drug powder is aseptically transferred to the clean presterilized powder filling machine hopper. It is agitated for uniform blend . 2) It is aseptically filled into the sterile vial using powder filling equipment with positive piston displacement filters or auger type filters .the vials are sealed with presterilized closures & aluminium crimp seals. 3) The fill weight controller is adjusted to the proper fill. filled vials are examined for particulate matter. 4) The containers are filled , stoppered, & sealed within sterile area & passed through into clean , nonsterile inspection area. 5) Filled containers are sampled at set intervals during the filling operation for checking fill, sterility, pyrogens, clarity, pH, colour and additional release test which are required for the drug. Formulatio for Small volume parenteral for constitution

Rx

Dosage 40 mg

Category active drug

Methylprednisolone sodium Succinate ( equivalent to Methyl prednisolone)

Sodium biphosphate anhydrous 1.6 mg Sodium phosphate dried Lactose ( hydrous) 17.4 mg 25 mg

Buffering agent Buffering agent Bulking agent

E) preparation of Aqueous Opthalmic solution productsThe formulation& preparation is same as discribe under A The principle difference between the two formulation is the typical parenteral product for injection contains bezyl alcohol, which is not normally use in opthalmic products. Benzalkonium chloride is used which is stable over a wide pH range & at hot condition but it produce salt when combined with negatively charged substances, it may migrate into plastic containers with the potential binding with the glue of the labeling . Other preservatives used are chlorobutanol, phenylmercuric nitrate & acetate, thiomersal, paraben. The formulator should be caution that some countries not permit use of certain preservatives, eg Japan. Sometimes EDTA is combined with benzalkonium chloride to potentiate the activity against Pseudominas. F) Preparation sterile Opthalmic suspension productsThe formulation & preparation is same as describe under B The formulator must take into consideration two things while using surface active agent: 1) the preservative activity may alter 2) the ocular irritation potential of surface active agent . They are known to bind with preservatives. The purpose of NaCMC is to help suspend the insoluble drug rather than to increase the contact time of the drug within the eye. G) preparaton of genetically Engineered or biotechnology products These products include monoclonal & other bio derived parenterals.

These formulation are processed using biotechnology, chemical processing & conventional scale-up , processing of sterile pharmaceutical products. Supplier, genotype & husbandry of animals used in in-vivo production must be controlled & documented There must be a step taken for prevention of viral, bacterial, fungal, & mycoplasmal contamination . The standard procedures of handling , compounding , sterilizing & aseptically subdividing the final product derived from monoclonal antibodies is same as used for preparaton of other small volume parenteral productions.

Formulation of bacterial vaccines/ Toxoids / viral vaccines( differes in source of antigen) Many bacterial vaccines are formulated as suspended cells, or filtrate sorbed onto adjuvants such as AlOH . The bacterial suspension are freeze dried for reconstitution before use or lyse the bacterial cells & then separate the solid via centrifuging. The vaccines are then packaged in such a manner that they can be reconstituted by diluent, ranging from water to buffer solution contained in a separate vial or prefilled syringe .the final product may be monovalent ( 1antigen) or multivalent . Although the specific steps in making vaccines & other biologicals such as immunoglobin or serum are geared toward separation , extraction, or purification of active immunological moieties from biological system, the basic aseptic processing steps and processing steps & equipment associated with the manufacture of sterile pharmaceuticals are the same . H) Preparation of liposomes & lipid productsThe processing of the lipid portion of the formulation must be accomplished at high temperature. Working at elevated temperatures is difficult to accomplish during aseptic processing high temperature can deleterious to the stability of the lipid. Sterilization by filteration and handling of organic solvent is problematic. Another challenge is the control of particle size of liposome droplets. Because preparation is administered by iv route. Particle size control is essential for proper biodistribution , targetting, & toxicity/ efficacy of the final formulation Phospholipids which contains unsaturated lipid chains are sensitive to oxidation & hydrolysis: particularly when the pH of aq portion is not neutral & the product is exposed to light. Precaution is taken while manufacturing process design & packaging design to minimize these condition and to maintain stability.

The stability of the phospholipid & active moiety or drug being encapsulated or entrapped in the liposome vesicle is of importance. One way to circumvent is a vial of drug product is freeze dried . The freeze dried drug is then constituted with a liposome dispersion.by altering the pH of the system, entrapment of the drug within the vesicle is accomplished at the time of use. Freeze drying of the liposomal product with suitable cryoprotection is another method of preserving stability of the system.

MANUFACTURING OF LVP
Introduction The USP defines large volume injections as products in containers labeled as containing more than 100 ml of a single-dose injection intended for administration by intravenous infusion. However, substantially the same technology is used to prepare other materials that do not contain preservatives such as irrigation solutions packaged in pour bottles, & bulk containers of water or aqueous Because these solutions are injected directly into the bloodstream, poured into open body cavities & surgicals areas , have direct contact with blood or are introduced into body cavities , they must be presented as sterile & non pyrogenic preparations. Although official particulate matter standards in the USP apply only to iv solutions , all of above preparations must be substantially free of particulate matter. Thus processing parameters should be such that the final product must be sterile nonpyrogenic &free of particulate.

Manufacturing process

Steps Of mfg.. 1. Selection of Vehicles 2. Selection of Raw Materials 3. Batch Mixing 4. Filtration 5. Containers & Closures 6. Filling 7. Sealing 8. Labeling 9. Final Product Testing 10.Process Validation Vehicles Water is used as vehicle for LVPs.

Water For Injection is water purified by distillation or by RO & contains no added substances. Water prepared by RO or Distillation , even though prepared in a properly designed , engineered , Installed & maintained system , is depended on designed , installation , & sanitation of the downstream side of the system , mainly piping , pumps , holding tanks , & controls exercised over the quality of the immediate environment . WFI is used as solvent in parentral solutions , whether terminally sterilized in the final container or prepared under the aseptic conditions & sterilized by appropriate filtration. Purified water , deionized water or potable water may be employed during early phase of the manufacturing processe-the preliminary cleaning of containers , parts , lines , & tanks . However , WFI is more routinely employed for final rinsing of these items & other contact surfaces. The choice of water for these early phases depends on the locale(place) & chemical & microbiological quality of the specific water.

Pretreatment Chlorination or ozone treatment for reducing microbial growth Pre filtration through depth filters(sand) to remove iron , suspended matter Flocculation for removal of suspended matters.(alum) Water softening by ion exchange pH adjustment to the range of 6.0 to 6.5 to reduce scale deposits. Deionization by ion exchange resins for more complete removal of ions from feedwater. Activated carbon bed for removal of chlorine & organics.

WFI production By Reverse Osmosis By Distillation

Raw materials Apart from WFI , there are many other substances that are added to the LVP

Carbohydrates such as Dextrose , Fructose , sucrose , maltose , dextran , etc. Amino acids Vegetable oil in case of Lipid emulsions Polyols like Glycerols(in irrigation solutions) , sorbitols(same) & mannitol(osmotic agent). Inorganic salts Acids & Bases for pH adjustment

Qualification & Stability Quality of the starting materials & solutes is critical to finished LVP products Appropriate parameters such as Insoluble particulate levels, Filterability of solutions containing the material, Physical attributes that may affect dissolution rate during production , & presence of contaminants & degradants

Stability consideration Organic materials are sensitive to heat Oils & Fats contains double bonds that may react with oxygen to form Peroxides Amino acids can be affected by heat , light , air & moisture content Anhydrous materials can take up moisture from environment Hydrated substance can deliquesce & effloresce Basic materials (NaOH) may absorb CO2 from air.

Receiving A specific area is usually dedicated as delivery point for the receipt of raw materials.

All raw materials must be inspected , identified , documented ,& sampled in accordance with SOP. All raw materials should be issued to production on a FIFO basis.

Storage/Quarantine areas All materials associated with the final production ( containers , closures , drug substance ) must be sampled , distributed to laboratory functions & tested for conformation to written specifications. caged area must be provided for label storage segregated into released & unreleased sections. This secured area should be locked when not in used. Similarly caged area should be provided for rejected raw materials.

Batch Mixing Most of all LVPs are simple solutions except Lipid emulsions. The solution mix tanks are filled with WFI & weighed solutes are added , with mixing to effect dissolution. The solution is then assayed to confirm the acceptability of the solutes concentrations. If not within the specification , the solution may be adjusted. If necessary by addition of WFI or any of solutes as required & then assayed.

Filtration Removal of foreign particles from the solution is must as these preparations are used in the body. This is achieved by filtration methods like Screen filters Depth filters Cake filters Membrane filters

Screen filters: A screen ( or sieve) filter can be visualized as a plate with holes in it. These holes enable particles with dimensions larger than the holes to be retained on the surface. Instead of a plate a woven sieve can also be used. The passage of fluid through the filter causes it to become less efficient as larger particles block the pores & reduce the flow,

Depth filters: The filtration medium consists of fibers or granular particulate solids arranged in a bed that allows the liquid to pass through. The particles are much more smaller than the interstices between the fibers of filter bed , so that they are not removed by straining or screening. The particles are trapped by gravitational , hydrodynamic , or electrical forces that are acting in the environment of the bed. a particle requires sufficient time of contact with the bed to leave the main stream of liquid flowing through the bed and approach close enough to solid granules to be captured by Vander walls forces at surfaces.

Cake filters: The properties of the cake are dependent on shape and size of rigid particles from which it is made foe eg: one of the most useful cakes can be made from diatomite that consists of irregularly shaped particles of around 100 micron diameter. A cake made from a suspension of diatomite removes submicrometer colloid particles with high efficiency from a product that is subsequently passed through the bed.

EVALUATION OF SMALL VOLUME PARENTERALS

1. Particulate Contamination Particulate contamination is defined as the unintentional presence of extraneous, mobile, undissolved substances, other than gas bubbles. Parenteral preparations are expected to be free from particles of approximately 50 m or more that can be observed by inspection with the unaided eye. For visible particles : For multiple dose injections, single dose small volume parenteral preparations and parenteral solutions constituted from sterile solids, the following method of visual assessment is adequate.

For sub-visible particles : Parenteral preparations in containers that are labelled as containing100 ml or more of a single-dose large volume injection

intended for administration by intravenous infusion should comply with the limits of sub-visible particles prescribed in this test. For sub-visible particles : Two methods are specified by IP 1. Involves the counting of particles viewed under a microscope. 2. Based on the count of particles causing light obscuration. Method 1. Microscopic particle count test This method is suitable for revealing the presence of particles the longest axis or effective linear dimension of which is 10 m or more.

Apparatus : The microscope is equipped with an ocular micrometer calibrated with an objective micrometer. The filter assembly consists of a filter holder of glass or metal and is connected to a vacuum source and a suitable membrane filter. The filter is black or dark grey in colour, is gridded or non-gridded and has a pore size of 1.0 m or less.

Method : Invert the container of the preparation under examination 20 times successively in order to mix the contents. Wash the outer surface of the container with a jet of particle free water and remove the closure carefully, avoiding contamination of the contents. Take the filter paper and Wash both sides of the membrane with a stream of particle-free water Place the membrane with the grid side up on the filter base and install the filtering funnel on the base without sliding the funnel over the membrane filter. Invert the unit and wash the inside of the funnel for about 10 seconds. Transfer to the filtration funnel the total volume of the pooled solution or of a single unit, allow to stand for a minute, apply the vacuum and filter. After filtration, remove the membrane filter with flat-ended forceps. Place the gridded surface of filter up in a Petri dish or slide and allow it to dry in air with the cover slightly a jar.

place the dish on the stage of the microscope. Count the number of particles that are equal to or greater than 10 m, the number of particles equal to or greater than 25 m and the particles equal to or greater than 50 m.

Method 2. Light obscuration particle count test Not suitable for preparations with reduced clarity or increased viscosity such as emulsions, colloids, liposomal preparations and products that produce air or gas bubbles when drawn into the sensor. Based on the principle of light blockage and capable of automatic counting and sizing of particles.

Method: Invert the container of the preparation under examination 20 times successively in order to mix the contents. Wash the outer surface of the container with a jet of particle free water and remove the closure carefully, avoiding contamination of the contents. Remove portions, each of not less than 5 ml, and count the number of particles equal to or greater than 10 m and 25 m.

Ignore the result obtained for the first portion, and calculate the average number of particles in the preparation under examination

1. Pyrogen Testing : Pyrogenic - means producing fever Pyrogens - fever inducing substances Having nature Endogenous (inside body) Exogenous (outside body)

Exogenous pyrogens mainly lipopolysaccharides bacterial origin, but not necessary

The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 minutes. Use healthy, adult rabbits of either sex, preferably of the same variety, weighing not less than 1.5 kg, fed on a complete and balanced diet and not showing loss of body weight during the week preceding the test.

House the animals individually in an area of uniform temperature ( 2), preferably with uniform humidity, and free from disturbances likely to excite them. Do not use animals for pyrogen tests more frequently than once every 48 hours. After a pyrogen test in the course of which a rabbits temperature has risen by 0.6 or more, at least 2 weeks must be allowed to elapse before the animals is used again.

GLASSWARE: All glassware, syringes and needles must be thoroughly Washed with water for injections and heated in a hot air oven At 250 for 30 minutes or at 200 for 1 hour. Treat all diluents And solutions for washing and rinsing of devices in a manner That will assure that they are sterile and pyrogen-free.

Preliminary Test (Sham Test) If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1 to 3 days before testing the substance under examination by injecting intravenously into them 10 ml per kg of body weight of a pyrogen-free saline solution warmed to about 38.5. Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after Injection of the solution being examined. Any animal showing a temperature variation of 0.6 or more must not be used in the main test. If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1 to 3 days before testing the substance under examination by injecting intravenously into them 10 ml per kg of body weight of a pyrogen-free saline solution warmed to about 38.5. Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6 or more must not be used in the main test.

Main Test Carry out the test using a group of three rabbits.

PREPARATION OF THE SAMPLE Dissolve the substance under examination in, or dilute with, pyrogen-free saline solution or other solution prescribed in the monograph. Warm the liquid under examination to approximately 38.5 before injection PROCEDURE Record the temperature of each animal at intervals of not more than 30 minutes, beginning at least 90 minutes before the injection of the solution under examination and continuing for 3 hours after the injection. Not more than 40 minutes immediately preceding the injection of the test dose, record the initial temperature of each rabbit, which is the mean of two temperatures recorded for that rabbit at an interval of 30 minutes in the 40-minute period. Rabbits showing a temperature variation greater than 0.2 between two successive readings in the determination of initial temperature should not be used for the test. In any one group of test animals, use only those animals whose initial temperatures do not vary by more than 1 from each other, and do not use any rabbit having a temperature higher than 39.8 and lower than 38. Inject the solution under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The amount of sample to be injected varies according to the preparation under examination and is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Record the temperature of each animal at half-hourly intervals for 3 hours after the injection. The difference between the initial temperature and the maximum temperature which is the highest temperature recorded for a rabbit is taken to be its response. When this difference is negative, the results is counted as a zero response.

INTERPRETATION OF RESULTS If the sum of the responses of the group of three rabbits does not exceed 1.4 and if the response of any individual rabbit is less than 0.6, the preparation under examination passes the test. If the response of any rabbit is 0.6 or more, or if the sum of the response of the three rabbits exceeds 1.4, continue the test using five other rabbits.

If not more than three of the eight rabbits show individual responses of 0.6 or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7, the preparation under examination passes the test.

Bacterial endotoxins To detect or quantify endotoxins of gram-negative bacterial origin Reagent: amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). The name of the test is also Limulus amebocyte lysate (LAL) test Mechanism of LAL the test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located with the crab's amebocyte blood cells endotoxins initiation of an enzymatic coagulation cascade proteinaceous gel

Commercially derived LAL reagents Bleeding adult crabs into an anticlotting solution Washing and centrifuging to collect the amebocyte Lysing in 3% NaCl Lysate is washed and lyophilized for storage Activity varies on a seasonal basis and standardization is necessary. Avoid endotoxin contamination Before the test: Test: equal portion of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube incubation at 37C, 1 hour remove the tube - invert in one smooth motion (180) - read (observe) the result interfering factors should not be present equipment should be depyrogenated the sensitivity of the lysate should be known

 LAL TEST

pass-fail test

Three different techniques: The gel-clot technique - gel formation The turbidimetric technique - the development of turbidity after cleavage of an endogenous substrate The chromogenic technique - the development of color after cleavage of a synthetic peptide-chromogen complex

6 methods with different steps of accuracy of LAL test results: Method A: gel-clot method: limit test Method B: gel-clot method: semi-quantitative test Method C: turbidimetric kinetic method Method D: chromogenic kinetic method Method E: chromogenic end-point method Method F: turbidimetric end-point method

In the event of doubt or dispute, the final decision is made upon Method A unless otherwise indicated in the monograph.

Gel-cloth technique (Methods A, B) Allows detection or quantification of endotoxins Clotting of the lysate in the presence of endotoxins. 1.Preparatory testing Confirmation of the labeled lysate sensitivity Tests for interfering factors

Limit test (method A) a firm gel - positive result. an intact gel is not formed - negative result. the interpretation of the results

Semi-quantitative test (method B) quantification of bacterial endotoxins in the test solution by titration to an end-point.

procedure is similar as in the limit test The results are expressed as concentration of endotoxin as less, equal or greater than (labeled lysate sensitivity).

Turbidimetric technique (Methods C, F) photometric test to measure the increase in turbidity End-point test (Method F): quantitative relationship between the endotoxin concentration and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. Kinetic test (Method C): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of turbidity development.

Chromogenic technique (Methods D, E) Measuring the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate End-point test (Method E): is based on the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period Kinetic test (Method D): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of color development

Advantages of LAL test Fast - 60 minutes vs. 180 minutes Greater Sensitivity Less Variability Much Less False Positives Much Less Expensive Alternative to Animal Model More accurate than other Can be performed in the pharmaceutical laboratory Specific for endotoxins of gram-negative origin Particularly useful for:

Radiopharmaceuticals and cytotoxic agents Products with marked pharmacological or toxicological activity in the rabbit (e.g. insulin) Blood products which sometime give misleading results in the rabbit Water for injection where LAL test is potentially more stringent and readily applied

Sterlity testing : The test for sterility is applied to pharmacopoeial articles that are required according to the Pharmacopoeia to be sterile The test must be carried out under aseptic conditions designed to avoid accidental contamination. For this purpose, a grade A laminar flow cabinet or an isolater is recommended. The following table gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to exclude contamination.