What is EU GMP Annex 1? The 2022 sterile manufacturing rules explained, clause by clause

EU GMP Annex 1 is the rulebook for making sterile medicines in Europe, and since its 2022 revision it is also the shared text of the PIC/S inspectorates. Anyone sourcing sterile peptide vials, lyophilization or fill-finish for a European, PIC/S or WHO-aligned market is in practice buying against Annex 1. This guide explains what the annex is, when the revision applied, how it is organised, and what the clauses that matter most for peptide and biosimilar vials actually say.

Quotes and clause numbers below come from the published text, Commission document C(2022) 5938 [1]. For a process-level walk-through of filling, see aseptic fill finish explained.

What is EU GMP Annex 1?

Annex 1, “Manufacture of Sterile Medicinal Products”, is an annex to the EU Guidelines for Good Manufacturing Practice in EudraLex Volume 4 [1]. Its scope covers “a wide range of sterile product types (active substance, excipient, primary packaging material and finished dosage form)”, from single units to large batches and from manual to highly automated processes [1]. It applies quality risk management (QRM) to the whole document “to ensure that microbial, particulate and endotoxin/pyrogen contamination is prevented in the final product” [1].

Two framing statements in section 1 explain how inspectors read it. First, “where specific limits or frequencies or ranges are specified, these should be considered as a minimum requirement”, because they reflect “historical regulatory experience of issues that have been identified and have impacted the safety of patients” [1]. Second, principles such as the contamination control strategy, cleanroom classification and gowning may be applied voluntarily to non-sterile products where contamination control matters, but a manufacturer that does so must document which principles it applied [1].

EMA states that “any manufacturer of medicines intended for the EU market must comply with EU good manufacturing practice (GMP) no matter where in the world they are located” [2]. For sterile products, that means Annex 1.

When did the revised Annex 1 come into force?

Milestone	Date	Source
Annex 1 first published (based on a PIC/S recommendation)	1971	[3]
Previous version in operation	1 March 2009 (vial capping provisions 1 March 2010)	[1]
PIC/S Annex 1 working group established	May 2014	[3]
Revised Annex 1 issued by the Commission	Dated 22 August 2022 (C(2022) 5938)	[1]
Revised Annex 1 in operation (EU and PIC/S)	25 August 2023	[1] [4]
Clause 8.123 (lyophilizer loading design and sterilisation frequency) in operation	25 August 2024	[1] [4]

PIC/S describes the 2022 text as the first full revision, restructuring the annex and “introducing the principles of Quality Risk Management to allow for the inclusion of new technologies and innovative processes” [3]. The revision was “driven jointly by PIC/S and the EMA Inspectors’ Working Group (IWG) on GMDP in close co-operation with the European Commission (EC) and the World Health Organization (WHO)”, and PIC/S Annex 1 is identical to the EU text apart from “very minor editorial differences” [3]. It entered into force as part of the PIC/S GMP Guide PE 009-17; PIC/S revised the guide again as PE 009-18 in September 2026 to update Annex 19, not Annex 1 [4]. WHO’s sterile GMP text, published in 2022 as Annex 2 of Technical Report Series 1044, was revised “in collaboration with the European Union and the Pharmaceutical Inspection Co-operation Scheme (PIC/S)” [5].

The Commission gave its reasons in the document itself: to reflect “changes in regulatory and manufacturing environments”, to let manufacturers use “enhanced process understanding by using innovative tools as described in the ICH Q9 and Q10 guidelines”, and to “remove ambiguity and inconsistencies” while accounting for “advances in technologies” [1].

How Annex 1 is organised

The 59-page annex has 11 sections [1]:

Section	Covers
1. Scope	Product types and use of the principles for non-sterile products
2. Principle	Key areas, QRM and the contamination control strategy
3. Pharmaceutical quality system	Sterile-specific PQS duties, including senior management oversight
4. Premises	Cleanroom grades, barrier technology (RABS, isolators), qualification
5. Equipment	Design and operation of equipment
6. Utilities	Water, steam, gases and vacuum
7. Personnel	Training, qualification and gowning by grade
8. Production and specific technologies	Aseptic and terminal sterilisation, filtration, lyophilization, form-fill-seal, single-use systems
9. Environmental and process monitoring	Monitoring limits, trending and aseptic process simulation
10. Quality control	Microbiology expertise, bioburden, sterility testing
11. Glossary	Definitions

The contamination control strategy (CCS)

The CCS is the core idea of the revision. Clause 2.3 says a CCS “should be implemented across the facility in order to define all critical control points and assess the effectiveness of all the controls (design, procedural, technical and organisational) and monitoring measures employed to manage risks to medicinal product quality and safety” [1]. The glossary defines it as “a planned set of controls for microorganisms, endotoxin/pyrogen and particles, derived from current product and process understanding” [1].

Clause 2.5 lists 16 elements a CCS should consider at minimum [1]:

1. Design of plant and processes, with documentation
2. Premises and equipment
3. Personnel
4. Utilities
5. Raw material controls, including in-process controls
6. Product containers and closures
7. Vendor approval (key component suppliers, sterilisation of components and single-use systems, critical service providers)
8. Management of outsourced activities and transfer of critical information between parties
9. Process risk management
10. Process validation
11. Validation of sterilisation processes
12. Preventive maintenance
13. Cleaning and disinfection
14. Monitoring systems, including the feasibility of alternative methods
15. Prevention mechanisms: trend analysis, investigation, root cause and CAPA
16. Continuous improvement

The CCS must be reviewed periodically and any change to the systems assessed for its impact “before and after implementation” (2.6) [1]. Clause 2.2 sets the order of priority: design first, then procedures, then monitoring, because “monitoring or testing alone does not give assurance of sterility” [1]. Clause 2.7 adds that “sole reliance for sterility or other quality aspects should not be placed on any terminal process or finished product test” [1]. Element 8 matters for anyone using contract fill-finish: the flow of critical information between sponsor and site is part of the site’s CCS.

Cleanroom grades and particle limits

Annex 1 keeps four grades (4.4). Grade A is “the critical zone for high-risk operations (e.g. aseptic processing line, filling zone, stopper bowl, open primary packaging or for making aseptic connections under the protection of first air)”. Grade B is the background for Grade A “where it is not an isolator”. Grades C and D handle less critical stages, act as isolator backgrounds, and can be used for “preparation/filling of terminally sterilised products” [1].

Table 1: total particles for classification

Grade	≥0.5 µm/m³ at rest	≥0.5 µm/m³ in operation	≥5 µm/m³ at rest	≥5 µm/m³ in operation
A	3,520	3,520	Not specified	Not specified
B	3,520	352,000	Not specified	2,930
C	352,000	3,520,000	2,930	29,300
D	3,520,000	Not predetermined	29,300	Not predetermined

Source: Annex 1 Table 1 [1]. Classification including 5 µm particles “may be considered where indicated by the CCS or historical trends” [1].

Table 5: total particles for routine monitoring

For monitoring (as opposed to classification), Table 5 adds 5 µm limits: 29 per m³ for Grade A at rest and in operation, 29 at rest and 2,930 in operation for Grade B [1]. Grade A must be monitored continuously for particles ≥0.5 and ≥5 µm “for the full duration of critical processing, including equipment assembly”, with a sample flow rate of at least 28 litres (1 ft³) per minute (9.16, 9.17) [1]. Occasional ≥5 µm counts in Grade A may be false counts, but “consecutive or regular counting of low levels may be indicative of a possible contamination event and should be investigated” [1].

Table 6: viable (microbial) action limits

Grade	Air sample (CFU/m³)	Settle plate, 90 mm (CFU/4 h)	Contact plate, 55 mm (CFU/plate)	Glove print, 5 fingers (CFU/glove)
A	No growth	No growth	No growth	No growth
B	10	5	5	5
C	100	50	25	Not applicable
D	200	100	50	Not applicable

Source: Annex 1 Table 6 [1]. The same air, settle plate and contact plate values apply during qualification (Table 2) [1].

Requalification of Grade A and B areas must happen at least every 6 months and of Grade C and D areas at least every 12 months (4.32) [1]. Unidirectional airflow should deliver a homogeneous air speed of 0.36–0.54 m/s (a guidance value) at the working position, “unless otherwise scientifically justified in the CCS” (4.30) [1].

Barrier technology: RABS and isolators

The 2022 text pushes barrier technology into the default. Clause 4.3 says RABS or isolators “are beneficial in assuring required conditions and minimizing microbial contamination associated with direct human interventions in the critical zone. Their use should be considered in the CCS. Any alternative approaches to the use of RABS or isolators should be justified” [1]. Clause 8.9 extends this to robotics and automation, citing “automated lyophilizer loading” as an example of eliminating direct human critical interventions [1].

The background required depends on the barrier: at least Grade B for RABS; generally at least Grade C for open isolators and at least Grade D for closed isolators, with the decision “based on risk assessment and justified in the CCS” (4.20) [1]. Isolator bio-decontamination “should be automated, validated and controlled within defined cycle parameters and should include a sporicidal agent” (4.22), and isolator glove integrity testing should generally happen “at a minimum frequency of the beginning and end of each batch or campaign” (4.21) [1]. Decontamination agents must be shown not to harm the product [1]; published work on vaporised hydrogen peroxide in an antibody filling isolator shows why, with protein oxidation observed above 100 ng/mL of hydrogen peroxide [6].

The reason for all this is the operator. A PDA analysis describes interventions as “a key source of microbiological contamination of sterile biopharmaceutical products, irrespective of the type of manufacturing system used” [7]. Annex 1 requires an authorised list of qualified interventions and full recording of each one in the batch record (8.16, 8.17) [1].

Sterile filtration and PUPSIT (clause 8.87)

If a product cannot be sterilised in its final container, Annex 1 requires filtration through a validated sterilising-grade filter “with a nominal pore size of a maximum of 0.22 µm” and aseptic filling into a previously sterilised container (8.79) [1]. The clause that drew most industry debate is 8.87: the sterilised filter assembly “should be verified by integrity testing before use (pre-use post sterilisation integrity test or PUPSIT)”, and a non-destructive post-use integrity test is required before the filter is removed from its housing [1].

Annex 1 accepts that “PUPSIT may not always be possible after sterilisation due to process constraints (e.g. the filtration of very small volumes of solution)”, and allows an alternative if “a thorough risk assessment has been performed” with controls covering filter sterilisation, the supply chain and product-specific risks of masking [1]. Laboratory data support the concern: with some clogging-prone fluids, post-use bubble point values rose enough to “camouflage a possible flaw in sterilizing filters” [8]. A 2025 PDA paper offers a risk framework for justifying alternatives, using the 2023 revision of ICH Q9 [9].

What Annex 1 says about lyophilization

For peptide and biosimilar vials, clauses 8.121 to 8.126 are the most relevant part of the annex [1].

Clause	Requirement (quoted or summarised from Annex 1)
8.121	Lyophilization “is a critical process step”; everything that can affect sterility is an “extension of the aseptic processing” from filling to completion of the cycle
8.122	Sterilisation of the lyophilizer, trays and vial support rings must be validated, and the hold time between sterilisation and use challenged in the APS; re-sterilise after maintenance or cleaning
8.123	Design to minimise operator intervention; dryers “manually loaded or unloaded with no barrier technology separation should be sterilised before each load”; for automated or closed-barrier systems, frequency justified in the CCS (in operation from 25 August 2024)
8.124	The vent filter is sterilised before each use and its integrity result forms part of batch release; “maximum permitted leakage of air into the lyophilizer should be specified and checked at the start of every cycle”
8.125	Trays checked regularly for damage or distortion
8.126	Loading pattern specified and documented; partially closed containers transferred “under grade A conditions at all times”; unsealed vials normally separated from operators by physical barriers; loading utensils sterile

Table 4 lists “loading of a lyophilizer” as a Grade A operation [1]. The aseptic process simulation for lyophilized products must cover “filling, transport, loading, a representative duration of the chamber dwell, unloading and sealing”, without actual freezing or boiling-over of the medium, and should replicate worst cases such as the longest loading time and the longest interval between lyophilizer sterilisation and use (9.33) [1]. The cycle science behind these controls is covered in our peptide lyophilization guide.

Aseptic process simulation, container closure and QC

Aseptic process simulation (section 9). Initial validation needs “at least three consecutive satisfactory simulation tests that cover all working shifts”; routine revalidation is normally “twice a year (approximately every six months) for each aseptic process, each filling line and each shift”; typically a minimum of 5,000 to 10,000 units are filled; and “the target should be zero growth” (9.38, 9.40, 9.46) [1]. Section 9 also states the simulation is not “the primary means to validate the aseptic process” (9.32) [1].

Container closure integrity. Containers closed by fusion with a volume of 100 ml or less need 100% integrity testing; vials sealed under vacuum must be tested for maintenance of vacuum before release and during shelf life; CCI validation must consider transport stresses; and “visual inspection is not considered as an acceptable integrity test method” (8.22, 8.24, 8.25) [1]. Annex 1 does not prescribe a method, and industry practice reflects that: a European white paper found “no gold standard for CCI test methods” [10].

Quality control (section 10). Bioburden must be tested on each batch of aseptically filled product, with limits set immediately before the final sterilising filter (10.3) [1]. Specifications should include microbial, particulate and endotoxin/pyrogen limits where monitoring or the CCS indicates a need (10.2) [1].

Annex 1 compared with FDA, WHO and India’s Schedule M

Topic	EU GMP Annex 1 (2022)	FDA aseptic guidance (2004)	WHO TRS 1044 Annex 2 (2022)	India revised Schedule M, Part II (2023)
Legal status	Part of EU GMP; applies to products for the EU market [1] [2]	Non-binding guidance interpreting 21 CFR 210/211 [11] [12]	WHO guideline, harmonised with EU/PIC/S [5]	Binding rules under the Drugs Rules, 1945 [13]
Contamination control strategy	Required, site-wide (2.3) [1]	Term not used [11]	Required, same structure as Annex 1 [5]	Term not used in Part II [13]
Grade A / critical zone, ≥0.5 µm/m³	3,520 [1]	Class 100 (ISO 5), 3,520 [11]	3,520 [5]	3,520 [13]
Grade A ≥5 µm limit for classification	Not specified [1]	Not tabulated	Not specified [5]	20 per m³ [13]
Filter integrity testing	PUPSIT expected unless justified, plus post-use test (8.87) [1]	Pre-use test “can be performed”; post-use test “should be routinely performed” [11]	PUPSIT and post-use test (8.87) [5]	Sterilised filter verified before use and confirmed immediately after use (6.11.2.7) [13]
Media fill frequency	About every 6 months per line and shift [1]	Semi-annual per line [11]	About every 6 months per line and shift [5]	Media fills required for aseptic products [13]

India’s revised Schedule M (G.S.R. 922(E), 28 December 2023) states that sterility of aseptically processed products is assured “by ‘media simulation’ or ‘media fill’ runs”, and its Part II classification table still uses the earlier format with 5.0 µm limits of 20 per m³ for Grade A and 29 per m³ for Grade B at rest [13]. An Indian site that is compliant with Schedule M is therefore not automatically compliant with the 2022 Annex 1; a European buyer should check the site against Annex 1 itself.

What Annex 1 means for sterile peptide products

Most peptide medicines are parenteral, and EMA’s 2025 synthetic peptides guideline applies the general sterilisation decision trees to them. It accepts sterile filtration plus aseptic processing only if terminal sterilisation “under the least stressful conditions (achieving the required SAL ≤ 10-6) causes significant degradation” [14]. EMA’s sterilisation guideline gives the underlying reason: with aseptic processing, “accidental contamination caused by inadequate technique cannot be reliably eliminated by monitoring and control” [15]. Once aseptic processing is justified, Annex 1 governs how it is done, and for lyophilized peptides that includes the dryer.

A short checklist for a sterile peptide project:

• Ask for the site’s CCS summary and how your product fits into it [1].
• Confirm the barrier technology (isolator or RABS) and the background grade [1].
• Review filter validation with your product and the PUPSIT position [1].
• Review recent APS results for the line, container and shifts you will use [1].
• For lyophilized products, review lyophilizer sterilisation frequency, leak-rate limits and the loading system [1].
• Check the site’s EU GMP certificate status in EudraGMDP, the EU database of manufacturing authorisations and GMP certificates [16].

How Winhycare Pharma works with Annex 1 projects

Winhycare Pharma offers lyophilisation and vial filling of peptides and biosimilars at WHO-GMP certified manufacturing partners in India, with projects managed from Hyderabad, and clients may bring their own peptide. The partner’s process details and the documentation relevant to a project’s target market, including how the partner’s process compares with Annex 1 expectations, are shared per project. European clients can start with lyophilisation for Europe; see also peptide lyophilisation services, biosimilar lyophilisation, or send a lyophilisation enquiry.

Research peptides supplied by Winhycare Pharma are for laboratory research use only. Not for human or veterinary use.