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Second Supplement to USP 34NF 29

Acceptance Table 3 Level A1 A2 Number Tested 6 6 Criteria No individual value exceeds 10% dissolved. Average of the 12 units (A1 + A2) is not more than 10% dissolved, and no individual unit is greater than 25% dissolved. Average of the 24 units (A1 + A2 + A3) is not more than 10% dissolved, and no individual unit is greater than 25% dissolved.

Physical Tests / 905 Uniformity of Dosage Units 5097

chapter apply to each drug substance being comprised in dosage units containing one or more drug substances, unless otherwise specified elsewhere in this Pharmacopeia. The uniformity of dosage units can be demonstrated by either of two methods, Content Uniformity or 3Weight3 Variation (see Table 1). The test for Content Uniformity of preparations presented in dosage units is based on the assay of the individual content of drug substance(s) in a number of dosage units to determine whether the individual content is within the limits set. The Content Uniformity method may be applied in all cases. The test for 3Weight3 Variation is applicable for the following dosage forms:
Solutions enclosed in unit-dose containers and into soft capsules; Solids (including powders, granules, and sterile solids) that are packaged in single-unit containers and contain no active or inactive added substances; Solids (including sterile solids) that are packaged in single-unit containers, with or without active or inactive added substances, that have been prepared from true solutions and freeze-dried in the final containers and are labeled to indicate this method of preparation; and Hard capsules, uncoated tablets, or film-coated tablets, containing 25 mg or more of a drug substance comprising 25% or more, by weight, of the dosage unit or, in the case of hard capsules, the capsule contents, except that uniformity of other drug substances present in lesser proportions is demonstrated by meeting the requirements for Content Uniformity.

A3

12

Buffer StageUnless otherwise specified 3in the individual monograph3, the requirements are met if the quantities of active ingredient dissolved from the units tested conform to Acceptance Table 4. Continue testing through the three levels unless the results of both stages conform at an earlier level. The value of Q in Acceptance Table 4 is 75% dissolved unless otherwise specified 3in the individual monograph3. The quantity, Q, 3specified in the individual monograph3 is the total amount of active ingredient dissolved in both the Acid and Buffer Stages, expressed as a percentage of the labeled content. The 5%, 15%, and 25% values in Acceptance Table 4 are percentages of the labeled content so that these values and Q are in the same terms.
Acceptance Table 4 Level B1 B2 Number Tested 6 6 Criteria Each unit is not less than Q + 5%. Average of 12 units (B1 + B2) is equal to or greater than Q, and no unit is less than Q 15%. Average of 24 units (B1 + B2 + B3) is equal to or greater than Q, not more than 2 units are less than Q 15%, and no unit is less than Q 25%.

(W1) (W2)

(W3)

(W4)

The test for Content Uniformity is required for all dosage forms not meeting the above conditions for the 3Weight3 Variation test.s1s2S (USP34)
Table 1. Application of Content Uniformity (CU) and Weight Variation (WV) Tests for Dosage Forms Dose & Ratio of Drug Substance 25 mg <25 mg and or 25% <25% WV CU WV CU CU CU WV CU

B3

12

Dosage Form

905 UNIFORMITY OF DOSAGE UNITS

Change to read:

Type Uncoated Coated Hard

Subtype Film Others Suspension, emulsion, or gel Solutions

Tablets

Capsules

Soft Single component

CU WV

CU WV

This general chapter is harmonized with the corresponding texts of the European Pharmacopoeia and the Japanese Pharmacopoeia. Portions of the general chapter text that are national USP text, and are not part of the harmonized text, are marked with symbols (33) to specify this fact. 3NOTEIn this chapter, unit and dosage unit are synonymous.3 To ensure the consistency of dosage units, each unit in a batch should have a drug substance content within a narrow range around the label claim. Dosage units are defined as dosage forms containing a single dose or a part of a dose of drug substance in each unit. The uniformity of dosage units specification is not intended to apply to suspensions, emulsions, or gels in unit-dose containers intended for external, cutaneous administration. The term uniformity of dosage unit is defined as the degree of uniformity in the amount of the drug substance among dosage units. Therefore, the requirements of this

WV Solution freezedried in final container Others

WV

Solids in single-unit containers

Multiple components

WV CU

WV CU

European Pharmacopoeia and Japanese Pharmacopoeia text not accepted by the United States Pharmacopeia: Alternatively, products listed in item (4) above that do not meet the 25 mg/25% threshold limit may be tested for uniformity of dosage units by Mass Variation instead of the Content Uniformity test if the concentration relative standard deviation (RSD) of the drug substance in the final dosage units is not more than 2%, based on process validation data and development data, and if there has been regulatory approval of such a change. The concentration RSD is the RSD of the concentration per dosage unit (w/w or w/v), where concentration per dosage unit equals the assay result per dosage unit divided by the individual dosage unit weight. See the RSD formula in Table 2.3s2S (USP34)
s1 3

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5098 905 Uniformity of Dosage Units / Physical Tests

Table 1. Application of Content Uniformity (CU) and Weight Variation (WV) Tests for Dosage Forms (Continued) Dose & Ratio of Drug Substance 25 mg <25 mg and or 25% <25%

Second Supplement to USP 34NF 29

sary to establish a correction factor to be applied to the results of the latter.

Solid Dosage Forms

Assay 10 units individually using an appropriate analytical method. Calculate the acceptance value (see Table 2).

Dosage Form Solutions in unit-dose containers 3and into soft capsules3 Others

Type

Subtype

Liquid sor Semi-Solids2S (USP34) Dosage Forms

WV CU WV CU
sAssay 10 units individually using an appropriate analytical method.s2S (USP34) Carry out the assay on the amount of wellmixed material that is removed from an individual container in conditions of normal use, and express the results as delivered dose. Calculate the acceptance value (see Table 2).

Change to read:

Calculation of Acceptance Value

Calculate the acceptance value by the formula:

CONTENT UNIFORMITY
Select not fewer than 30 units, and proceed as follows for the dosage form designated. Where different procedures are used for assay of the preparation and for the Content Uniformity test, it may be neces-

in which the terms are as defined in Table 2.

Table 2 Variable Definition Mean of individual contents (1, 2, , n), expressed as a percentage of the label claim Individual contents of the units tested, expressed as a percentage of the label claim Sample size (number of units in a sample) Acceptability constant Sample standard deviation Conditions Value

X 1, 2, , n

n k s

If n = 10, then k = If n = 30, then k =

2.4 2.0

RSD

M (case 1) to be applied when T 101.5

Relative standard deviation (the sample standard deviation expressed as a percentage of the mean) Reference value

100s/X

If 98.5% X 101.5%, then If X <98.5%, then If X >101.5%, then

M (case 2) to be applied when T >101.5

Reference value

If 98.5 X T, then If X <98.5%, then If X >T, then

Acceptance value (AV)

M = X (AV = ks) M = 98.5% (AV = 98.5 X + ks) M = 101.5% (AV = X 101.5 + ks) M=X (AV = ks) M = 98.5% (AV = 98.5 X + ks) M = T% (AV = X T + ks) General formula:

(Calculations are specified above for the different cases.)

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Second Supplement to USP 34NF 29

Physical Tests / 905 Uniformity of Dosage Units 5099

Table 2 (Continued)

Variable L1 L2

Definition Maximum allowed acceptance value Maximum allowed range for deviation of each dosage unit tested from the calculated value of M

Conditions

Value L1 = 15.0 unless otherwise specified L2 = 25.0 unless otherwise specified

On the low side, no dosage unit result can be less than [1(0.01)(L2)]M, while on the high side, no dosage unit result can be greater than [1 + (0.01)(L2)]M. (This is based on an L2 value of 25.0.)

Target content per dosage unit at the time of manufacture, expressed as a percentage of the label claim. sUnless otherwise stated, T is 100.0%, or T is the manufacturers approved target content per dosage unit.s2S (USP34)
3

WEIGHT3 VARIATION

Liquid Dosage Forms

Accurately weigh the amount of liquid that is removed from each of 10 individual containers in conditions of normal use. If necessary, compute the equivalent volume after determining the density. Calculate the drug substance content in each container from the mass of product removed from the individual containers and the result of the Assay. Calculate the acceptance value.

Carry out an assay for the drug substance(s) on a representative sample of the batch using an appropriate analytical method. This value is result A, expressed as percentage of label claim (see Calculation of Acceptance Value). Assume that the concentration (weight of drug substance per weight of dosage unit) is uniform. Select not fewer than 30 dosage units, and proceed as follows for the dosage form designated.

Uncoated or Film-Coated Tablets

Accurately weigh 10 tablets individually. Calculate the content, expressed as percentage of label claim, of each tablet from the 3weight3 of the individual tablet and the result of the Assay. Calculate the acceptance value.

Calculation of Acceptance Value

Calculate the acceptance value as shown in Content Uniformity, except that the individual contents of the units are replaced with the individual estimated contents defined below.
1, 2, , n w1, w2, , wn A = = = individual estimated contents of the units tested, where i = wi A/W individual 3weights3 of the units tested content of drug substance (% of label claim) obtained using an appropriate analytical method mean of individual 3weights3 (w1, w2, , wn)

Hard Capsules
Accurately weigh 10 capsules individually, taking care to preserve the identity of each capsule. Remove the contents of each capsule by a suitable means. Accurately weigh the emptied shells individually, and calculate for each capsule the net 3weight3 of its contents by subtracting the 3weight3 of the shell from the respective gross 3weight3. Calculate the drug substance content of each capsule from the 3net weight3 of the individual capsule 3content3 and the result of the Assay. Calculate the acceptance value.

Change to read:

Soft Capsules
Accurately weigh 10 intact capsules individually to obtain their gross 3weights3, taking care to preserve the identity of each capsule. Then cut open the capsules by means of a suitable clean, dry cutting instrument such as scissors or a sharp open blade, and remove the contents by washing with a suitable solvent. Allow the occluded solvent to evaporate from the shells at room temperature over a period of about 30 minutes, taking precautions to avoid uptake or loss of moisture. Weigh the individual shells, and calculate the net contents. Calculate the drug substance content in each capsule from the 3weight3 of product removed from the individual capsules and the result of the Assay. Calculate the acceptance value.

CRITERIA
Apply the following criteria, unless otherwise specified.

Solid, sSemi-Solid,s2S (USP34) and Liquid Dosage Forms

The requirements for dosage uniformity are met if the acceptance value of the first 10 dosage units is less than or equal to L1%. If the acceptance value is > L1%, test the next 20 units, and calculate the acceptance value. The requirements are met if the final acceptance value of the 30 dosage units is L1%, and no individual content of 3any3 dosage unit is less than [1 (0.01)(L2)]M nor more than [1 + (0.01)(L2)]M 3as specified3 in the Calculation of Acceptance Value under Content Uniformity or under 3Weight3 Variation. Unless otherwise specified, L1 is 15.0 and L2 is 25.0.

Solid Dosage Forms Other Than Tablets and Capsules

Proceed as directed for Hard Capsules, treating each unit as described therein. Calculate the acceptance value.

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5100 1027 Flow Cytometry / General Information

Second Supplement to USP 34NF 29

General Chapters
General Information
Add the following:

1027 FLOW CYTOMETRY

INTRODUCTION

Flow cytometry is an analytical method that plays a critical role in the quantitative and qualitative assessment of cell populations in patient and cellular product samples. The power of flow cytometry lies in its ability to rapidly and reliably analyze multiple attributes of individual cells within heterogeneous cell populations. Despite the value of flow cytometry data, method validation is challengingperhaps more so than for other analytical methodsbecause of errors and artifacts from multiple sources. Although flow cytometric methods can also be used to sort and isolate cells as part of the manufacturing process for cell- and tissue-based biological products, the scope of this chapter is limited to the use of flow cytometry as an analytical method. This chapter presents the technical aspects of the method, including instrumentation, sample handling and staining, and data analysis. Sources of error are considered in the context of technical features, as well as in the discussion of quality control, quality assurance, and standardization. Finally, current applications and assay troubleshooting principles are presented. For additional information on the basics and practical aspects of flow cytometry, see the current edition of Practical Flow Cytometry (Shapiro, 2003). Flow cytometry is widely used to characterize cell and tissue-based products, but most assay methods are not yet standardized. In addition to issues related to technical complexity, there are also challenges to standardization of flow cytometric methods for specific product classes or types related to the heterogeneous nature of these products, even among those with similar manufacturing processes and clinical uses. Current and future innovations in instrumentation, analytic reagents, analytic algorithms, and automation are likely to improve the technologys capabilities but are unlikely to eliminate challenges (e.g., bioassay, identification tests, and other applications).

that individual cells can be observed or interrogated for characteristics such as size, granularity, and presence of surface membrane or intracellular antigens or molecules. The cells are suspended in fluid in which movement is controlled by the size and configuration of tubing, chambers, and pumps specific to the flow cytometry instrument. The pattern of light signals produced from the laser lights interaction with the cells is captured by a detection system, also specific to the instrument, and the detected signals are transformed into data elements that can be analyzed and combined with data from other cells in a given sample. Data from a cell suspension can then be expressed and presented in one-, two-, or three-dimensional visual formats, or in numerical formats, to characterize the cellular sample and its subpopulations both qualitatively and quantitatively.

Flow Cytometry Instrumentation

Flow cytometers, which incorporate fluidic, optical, and electronic signal processing elements, are described below. FLUIDICS The fluidics system moves a bulk mixture of cells so that a stream of single cells is formed. Within the flow cytometer, the single-cell suspension passes through a confined region where each cell is sequentially illuminated by a uniform light source at the observation point (interrogation point). Most instruments use a flow chamber (flow cell) that, after the cell sample is drawn into the sample injection tip, combines the cells with isotonic sheath fluid, using a conical nozzle assembly that is geometrically designed to produce a laminar flow of fluid (Figure 1). The fluid outlet nozzle typically has an orifice of 50250 m in diameter through which fluid exits at a high flow rate. Differential pressures between the sample stream of cells (lower pressure) and the sheath stream (higher pressure) draw the cells/particles out into a confined stream. The resulting coaxial stream within a stream is highly efficient, and the sample stream at the observation point is typically only slightly larger than the cells or particles contained within. At least one manufacturer uses an alternative approach in which the coaxial stream strategy is replaced by the use of microcapillaries to focus and direct the cells. The light signal deflected or emitted by the cell is then measured and analyzed.

PRINCIPLES OF OPERATION, METHODS, QUALITY, AND STANDARDIZATION

The process of flow cytometry requires that cells move past a fixed light source consisting of one or more lasers so

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