SOP: Bioburden Testing and In-Process Bioburden Limits

This is a ready-to-use SOP for bioburden testing and the in-process bioburden control points that depend on it. Replace every <<FILL: ...>> placeholder with your own specifics, set your document numbers and dates, and route it through your normal document control, review, and approval. A worked filled specimen follows the template. The compendial chapters referenced here are copyrighted; this procedure describes them in its own words and states only numeric conditions. Read the current chapter text and confirm your own filed commitments before you rely on any of it. This is educational reference content, not regulatory advice.

Document control header

1. Purpose

This procedure defines how <<FILL: COMPANY NAME>> samples, tests, and reports bioburden, and how in-process bioburden limits are set, applied, and acted on. The objective is that every reported count is representative of the material at the control point it was taken from, was produced by a method shown to recover organisms from that matrix on media shown to grow them, and is assessed for what the organisms are, not only how many there were.

2. Scope

This procedure applies to bioburden testing of in-process bulk solutions, raw materials, components, intermediates, and finished nonsterile products at the sites named in the header.

It does not cover:

• Sterility testing, governed by <<FILL: SOP-ID>>. See sterility testing (USP <71>).
• Environmental monitoring, governed by the environmental monitoring SOP.
• Water system sampling, governed by the water system sampling SOP.
• Endotoxin testing, governed by the bacterial endotoxin testing SOP.
• Derivation of alert and action limits, performed on the bioburden alert and action limit derivation form.

3. Responsibilities

4. Definitions

• TAMC: total aerobic microbial count, the count of viable aerobic bacteria recovered on soybean-casein digest agar.
• TYMC: total combined yeasts and moulds count, the count of viable fungi recovered on Sabouraud dextrose agar.
• Control point: a defined place and time in the process where a bioburden sample is taken because the result informs a decision.
• Method suitability: the demonstration that the chosen recovery method actually recovers organisms from the specific matrix being tested.
• Objectionable organism: an organism that presents a hazard for the route of administration, can proliferate in the product, or compromises the dosage form, whether or not it is named in a compendial chapter and whether or not the count limit was met.

5. Procedure

5.1 Choosing the recovery method

USP <61>, Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests, describes three recovery methods. The method is selected per product and matrix during method suitability, recorded in the test method, and not changed at the bench.

For the sterile filtration control point in section 5.5, membrane filtration is generally the only method that can test the required volume, and the volume is part of the limit.

5.2 Method suitability

No bioburden result is meaningful until the method has been shown to recover organisms from that matrix.

1. Inoculate the prepared sample with not more than approximately 100 CFU of each challenge organism.
2. Run a parallel inoculum control without product.
3. Recover and count by the intended method.
4. Acceptance: the count recovered from the product preparation stays within a factor of two of the parallel control.

Standard challenge panel:

Where recovery falls outside the factor of two, the product is inhibitory. Modify the method (increase the neutraliser, increase the rinse volume, increase dilution, or move to membrane filtration with rinsing) and repeat. Neutralisers commonly used include lecithin and polysorbate; the neutralisation itself is demonstrated, not assumed.

Repeat method suitability when the formulation changes materially, when the manufacturing process changes in a way that could affect the matrix, and at the frequency stated in <<FILL: SOP-ID for periodic method review>>.

5.3 Media and reagent control

A count is only as good as the media that produced it.

1. Growth promotion testing. Every lot of prepared or purchased media is challenged with the relevant organism panel at not more than approximately 100 CFU and shown to support recovery, comparably to a previously approved lot, before or contemporaneously with its use in testing. A lot whose growth promotion was never confirmed leaves every negative result produced on it unsupported, and this is a recurring citation.
2. Media negative control. Incubate uninoculated units from each lot alongside the test to demonstrate the media were not contaminated.
3. Diluents and rinse fluids are qualified for sterility and for absence of inhibitory or promoting effects, and are lot-controlled.
4. Record every media lot, diluent lot, and rinse fluid lot on the test record, with expiry.
5. Store and use media within the labelled conditions and shelf life, and record any excursion.

5.4 Incubation and counting

1. Do not shorten the incubation period to meet a schedule. Slow growers and stressed organisms are exactly the ones a shortened incubation loses.
2. For the plate count method, count plates giving a countable range, typically not more than 250 colonies for TAMC and not more than 50 for TYMC, on the most appropriate dilution.
3. Multiply the count by the dilution factor and express per gram, per mL, per unit, or per the stated volume, as the specification requires.
4. Where the specification is expressed per 100 mL, report per 100 mL against the volume actually tested. Testing 1 mL and multiplying by 100 does not produce the same test and is not an acceptable substitute.
5. Record the count for every plate, including zeros, and retain the plates until the result is reviewed or per <<FILL: retention period>>.

5.5 The sterile filtration control point

For an aseptically filled product, the most consequential bioburden number is the load presented to the sterilising-grade filter.

1. Sample the bulk solution immediately before sterile filtration, at the point defined in the batch record, after the last operation that could change the load.
2. Test a 100 mL sample by membrane filtration, so the method has the sensitivity to detect at the limit.
3. Apply the limit stated in the product specification. The EMA guideline on the sterilisation of the medicinal product, active substance, excipient and primary container (EMA/CHMP/CVMP/QWP/850374/2015, which came into effect on 1 October 2019) describes not more than 10 CFU per 100 mL TAMC as acceptable in most situations for the bulk immediately before sterile filtration. Confirm what your own filing commits to, because that governs for your product.
4. Where a pre-filter is present only as a precaution rather than because the unfiltered bulk carries a higher load, the same limit applies before the pre-filter as well.
5. Where a starting material is known to carry inherent microbial contamination, a higher limit before pre-filtration may be acceptable, provided it is demonstrated that the first filter brings the load to not more than 10 CFU per 100 mL before the final filtration.
6. The same guideline addresses pre-sterilisation bioburden for other cases: a maximum of 100 CFU per 100 g or 100 CFU per 100 mL is described as acceptable without further justification for parenteral finished product formulations, and 10 CFU per gram or per mL for active substances and for finished products not used parenterally. Read the current text for the operative wording and confirm what applies to your product.

Two points to hold onto. The sample volume is part of the limit, not an incidental detail. And the limit is a specification at a control point, not a target to approach; a process routinely delivering 8 CFU per 100 mL against a limit of 10 is telling you something about upstream control, not comfortably passing.

Related reading: sterilising-grade filtration validation.

5.6 USP <1111> is not the in-process limit

USP <1111>, Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations, assigns acceptance limits by dosage form and route for finished nonsterile products. It is not the limit for an in-process control point, and applying it as one is a category error that appears in real deviation records.

Do not carry a remembered <1111> figure into any decision. Read the current chapter for the category that applies. Sterile products are not tested against <1111> at all; they are tested by sterility test and must show no recovery.

5.7 Sample timing, volume, and hold time

Timing is the variable that most often makes a count unrepresentative, and it matters more than the count itself.

1. Sample at the time and place defined in the batch record. A sample taken 40 minutes early, before the last addition, is not a sample of the material that was filtered.
2. Record the actual sampling date and time on the sample label and in the batch record.
3. Draw the full specified volume. A short sample is a deviation, not a smaller test.
4. Deliver to the laboratory and begin testing within the validated maximum hold time, <<FILL: e.g. not more than 4 hours at 2 to 8 C>>. Holding lets organisms grow or die, so the count drifts away from the true load at the control point in an unpredictable direction.
5. Store within the validated condition during transport, and record the storage condition and the time testing began.
6. A sample exceeding the hold time is recorded as a deviation, assessed for impact, and not simply tested late and reported as if the timing were compliant.
7. Establish the maximum hold time by study, per matrix, rather than by convention.

5.8 Objectionable organism assessment

A count within limits does not close the question.

1. Identify recovered isolates to the taxonomic level defined in <<FILL: SOP-ID for microbial identification>>, and at minimum where the count exceeds an alert limit, where the organism appears atypical, or where the control point is the sterile filtration bulk. See the microbial identification SOP.
2. Assess each identified isolate for objectionability against the route of administration, the ability to proliferate in the product, and the effect on the dosage form. Record the assessment on the objectionable organism determination form.
3. An organism can be objectionable even where the count limit was met and even where it is not named in USP <62>, Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. The compendial named list is a floor, not the whole question. Burkholderia cepacia complex in aqueous products is the standard example and has driven recalls.
4. Where an isolate is assessed as objectionable, raise a deviation immediately regardless of the numeric result, quarantine the affected material, and route per <<FILL: SOP-ID for deviations>>.
5. Retain isolates for <<FILL: retention period>> so a later investigation can compare them against environmental and water system isolates. See microbial identification and EM excursions.

5.9 Excursions and out-of-specification results

1. An alert limit excursion is an early warning. Notify the supervisor and the process owner, review recent results for a trend, and record the review. It is not automatically a deviation unless your procedure defines it as one.
2. An action limit excursion triggers the defined response in the limit derivation record: investigation, notification, and a decision on the affected material.
3. A result at or above the specification or regulatory limit is an out-of-specification result. Report it the same working day, quarantine the material, and route per <<FILL: SOP-ID for OOS>>. See the OOS investigation process.
4. Do not retest into compliance. A repeat test without an approved investigation and an assignable cause is not evidence.
5. Do not average a failing plate with a passing one, and do not exclude a plate without a documented assignable cause such as a recorded laboratory error.
6. For an excursion at the sterile filtration control point, assess the filtration step, the water system, hold times, upstream materials, and the environmental monitoring record for the same period.

6. Acceptance criteria

A reportable bioburden result is acceptable when all of the following are true.

• The sample was taken at the defined control point, at the defined volume, and tested within the validated hold time, with the actual times recorded.
• The recovery method was the one qualified for that matrix, and method suitability for that matrix is current.
• Every media lot, diluent lot, and rinse fluid lot used had current growth promotion evidence and an acceptable media negative control.
• Incubation conditions and durations were as stated in section 5.4, unshortened.
• The count was expressed on the same basis as the limit, using the volume actually tested.
• Isolates requiring identification were identified and assessed for objectionability.
• Any excursion or out-of-specification result was routed before any repeat testing.

7. References

21 CFR 211.84 (testing of components), 211.110 (in-process control), 211.113 (control of microbiological contamination), 211.165, 211.167. USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. USP <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations (finished nonsterile products only). USP <1227> Validation of Microbial Recovery from Pharmacopeial Articles. Ph. Eur. 2.6.12 and 2.6.13 (harmonised equivalents). EMA/CHMP/CVMP/QWP/850374/2015, Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container, effective 1 October 2019. EU GMP Annex 1, Manufacture of Sterile Medicinal Products. ICH Q9, Quality Risk Management.

Confirm the current version and clause numbers of each reference before issue.

8. Record generated: bioburden test record

9. Revision history

10. Approvals

Filled specimen

The following shows the record completed for an example bulk bioburden sample at the sterile filtration control point, and a second example showing an excursion. Company, product, and numbers are illustrative; replace them with your own.

Example 1: routine result, within limits.

Example 2: action limit excursion.

Sample BB-2026-0788, same control point, TAMC 8 CFU per 100 mL against an alert limit of 5 and an action limit of 7. The count was below the specification of 10, so it was not an out-of-specification result, but it exceeded the action limit and triggered the defined response.

The isolates were identified as Ralstonia species. That identification changed the assessment: a water-associated Gram-negative organism at the sterile filtration control point pointed upstream rather than at the sample. A deviation was raised on the identification, not on the count. The investigation reviewed the purified water loop sampling results for the preceding 30 days, found a rising trend at one point of use, and the corrective action was a sanitisation and a change to the loop sampling frequency. The batch was assessed and released on the basis that the load was well within the validated filter challenge, with the rationale recorded.

The sequence is the point. A count of 8 against a limit of 10 could have been recorded as a pass. What made it useful was that the organism was identified, the identification was assessed against the route and the control point, and the signal was followed upstream.

Common inspection findings this SOP prevents

• USP <1111> acceptance limits applied to an in-process control point, in place of the specification and the applicable sterilisation guideline expectation.
• A 1 mL sample tested and multiplied to report against a limit expressed per 100 mL, so the method never had the sensitivity the limit assumes.
• Bioburden sampled at a convenient moment rather than immediately before sterile filtration, so the result does not describe the load that was actually filtered.
• Samples held well beyond a validated hold time, or held with no validated hold time at all, and the results reported as if timing were controlled.
• Media lots used with no growth promotion evidence, or growth promotion performed long after the media were used in testing.
• Incubation cut short to fit a release schedule, so slow growers were never counted.
• Organisms recovered, counted, and never identified, so an objectionable organism assessment was impossible.
• An isolate assessed as acceptable purely because the numeric count was within limits.
• A method used on a matrix for which suitability was never demonstrated, or never repeated after a formulation change.
• Failing results retested until a passing one appeared, with no assignable cause and no investigation.

How to adapt this SOP

1. Set your document number, owner, and effective date in the header, and list your actual control points and matrices in scope in section 2.
2. Record the qualified recovery method per matrix in your test methods, and reference the method suitability report in each one.
3. Fill in your real specification, alert limit, and action limit for each control point, and keep them on the limit derivation form so the basis travels with the number.
4. Establish and state the validated hold time per matrix in section 5.7 rather than carrying a generic figure.
5. Set the identification trigger in section 5.8 to match your risk assessment, and point the cross-references to your real identification, deviation, and OOS procedures.
6. Confirm the EMA guideline expectations and your own filed commitments before setting the sterile filtration limit, since the filing governs for your product.
7. Read the current compendial chapters for the incubation conditions and the finished product acceptance criteria before issue rather than relying on any summary, including this one.