Principle of Autoclave and SOP for Pharmaceutical Sterilization

An autoclave is a critical piece of equipment in pharmaceutical manufacturing and microbiology laboratories that achieves sterilization through moist heat under controlled pressure. The principle underlying autoclave sterilization—moist heat sterilization—works by generating saturated steam at temperatures of 121–134°C and pressures of 15–30 psi, which denatures microbial proteins and destroys nucleic acids. This article provides comprehensive coverage of autoclave sterilization principles, validated operational procedures, regulatory compliance frameworks, and best practices for pharmaceutical quality assurance professionals, laboratory technicians, and students.

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What is an Autoclave? A Complete Definition

An autoclave is a precision-engineered, sealed chamber used to sterilize equipment, materials, and media by subjecting them to pressurized saturated steam at high temperatures. In pharmaceutical manufacturing, autoclaves are essential for sterilizing drug components, containers, raw materials, and equipment to ensure Sterility Assurance Level (SAL) of 10⁻⁶ or better—meaning no more than one contaminated unit per million processed items.

Key Function: Autoclaves eliminate all viable microorganisms—bacteria, viruses, spores, fungi, and prions—through protein denaturation and cell wall disruption caused by saturated steam penetration.

Why Autoclaves Matter in Pharma:

• Regulatory requirement for injectable medications (21 CFR 211.113)
• Critical for Sterile Area Classification (ISO 14644) compliance
• Essential validation step for drug product release
• Mandatory for medical device sterilization (ISO 11135, ISO 11137 series)

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The Scientific Principle of Autoclave Sterilization

Understanding Moist Heat Sterilization

Autoclave sterilization operates on the principle of moist heat sterilization, which differs fundamentally from dry heat and other methods. The process works through three primary mechanisms:

1. Protein Denaturation & Coagulation

Saturated steam at elevated temperatures (121–134°C) penetrates microbial cell walls and denatures essential proteins, causing irreversible structural changes. This coagulation of ribosomal proteins and enzymes is the primary killing mechanism. The presence of moisture facilitates this process more efficiently than dry heat because water molecules act as a carrier, accelerating heat transfer into bacterial spores.

2. Nucleic Acid Degradation

The combination of heat and moisture causes breakage of hydrogen bonds in DNA and RNA, preventing microbial replication even if the organism initially survives.

3. Cell Wall Disruption

Steam pressure (typically 15–30 psi above atmospheric pressure) forces steam molecules into bacterial cell walls, causing osmotic stress and mechanical rupture of the cell membrane.

Why Steam Sterilization is Superior to Dry Heat

Factor	Moist Heat (Autoclave)	Dry Heat Oven
Penetration	Rapid (steam diffuses quickly)	Slow (air is poor conductor)
Temperature Required	121–134°C for 15–30 min	160–170°C for 1–2 hours
Spore Destruction	✅ Highly effective	⚠️ Requires much higher temps
Material Compatibility	✅ Better for most pharma materials	❌ Damages heat-sensitive items
Energy Efficiency	✅ Lower temperature, faster cycle	❌ Higher energy consumption
Equipment Damage	Lower risk	Higher risk (oxidation, degradation)

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Standard Operating Procedure (SOP) for Autoclave Operation

1. Pre-Operation Checklist (Pre-Autoclave Phase)

A. Equipment Inspection

•  Verify autoclave chamber interior is clean and free of debris
•  Inspect door gasket for cracks, deterioration, or residue
•  Check water level in reservoir (if gravity-fed system) and top up if necessary
•  Test drain valve for proper function and absence of blockages
•  Verify temperature and pressure gauge calibration (if not digital)
•  Confirm steam supply is turned on and pressure is stable at 0–5 psi
•  Inspect and document any visible rust or corrosion

B. Load Preparation & Assembly

•  Ensure all items to be sterilized are clean and free of organic matter
•  Remove metal caps from bottles; leave caps loose or use vented caps for media
•  Place indicators (chemical or biological) inside the load for validation
•  Wrap items in autoclavable wrapping or use perforated containers (Petri dishes, flasks)
•  Do NOT overload the chamber—maintain at least 1 inch clearance on all sides
•  Position larger items on lower racks; smaller items above
•  For liquid media: use Erlenmeyer or dedicated containers; fill only 2/3 full to prevent boiling over
•  For pipette tips and other small items: use dedicated autoclavable containers with vents

C. Load Documentation

•  Record load number, contents, and date on load log
•  Document number of containers, estimated weight
•  Photograph the loaded chamber for compliance records (optional but recommended)

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Autoclave Cycle Operation (Exposure Phase)

Standard Sterilization Cycles for Pharmaceutical Use

Cycle Type	Temperature	Pressure	Duration	Best Use	F₀ Value Target
Gravity Displacement (Standard)	121°C	15 psi	15–30 min	Non-wrapped items, solid waste, media	10–15 min
Pre-Vacuum (High Speed)	132–134°C	20–30 psi	3–10 min	Wrapped instruments, packages, dense loads	15–20 min
Liquid Cycle	121°C	15 psi	30–40 min	Media, solutions; slow exhaust to prevent boiling	10–15 min
Low-Temperature Prion Cycle	134°C	30 psi	18 min	Prion-contaminated materials (CJD risk)	18+ min

Selection Criteria: Choose cycle based on:

• Material composition (glass, metal, plastic compatibility)
• Load density (tightly packed = longer cycle needed)
• Humidity sensitivity of contents
• Regulatory requirements (pharma vs. microbiology lab)

Step-by-Step Cycle Execution

1. Close Chamber Door:
   • Ensure door seals properly; resistance should be felt when closing
   • Verify indicator light confirms door lock engagement
   • Do NOT force door; resistance confirms proper gasket seating
2. Select Cycle Program:
   • Modern autoclaves: Select program from digital touchscreen
   • Older models: Adjust manual dials to desired temperature and pressure
   • Always verify settings on display before pressing START
3. Initiate Sterilization:
   • Press START button
   • Monitor initial pressure rise (should reach target within 5–10 minutes)
   • Temperature ramping phase: 3–5 minutes to reach setpoint
   • Exposure phase: Maintain constant temperature and pressure for specified duration
4. Monitor Running Cycle:
   • Observe temperature and pressure gauges continuously for first 2–3 minutes
   • Verify no unusual sounds (hissing, banging, or pressure release)
   • Check that exhaust steam is flowing steadily from vent port
   • Modern autoclaves: System will alarm if parameters deviate >2°C or ±2 psi
5. Exhaust Phase:
   • After exposure time expires, autoclave automatically begins controlled venting
   • Pressure decreases gradually (rapid depressurization can damage containers)
   • Slow exhaust for liquids: 5–10 minutes to prevent boiling over and media loss
   • Normal exhaust for solids: 2–3 minutes
6. Chamber Cool-Down:
   • Do NOT open door until temperature falls below 80°C
   • Allow 20–30 minutes for natural cooling (or per manufacturer protocol)
   • Rushing cool-down risks:
     • Water droplet condensation inside packages
     • Burn injuries to operator
     • Compromised sterilization validation (premature opening = air leakage)

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Post-Operation Procedures (Post-Autoclave Phase)

A. Unloading & Material Handling

•  Verify cycle completion on display; confirm all parameters in green/pass zone
•  Wait minimum 20 minutes after cycle end before opening door (optional but safer)
•  Carefully open door—watch for residual steam
• Inspect chemical or biological indicators for color change:
  • Green/black color shift = Successful sterilization ✅
  • No color change = Sterilization failure ⚠️ (Quarantine load)
•  Allow 10–15 more minutes for items to cool before removing from chamber
•  Handle items with care—they remain hot and can cause burns
•  Place items in designated cool-down area (not on cold surfaces to prevent thermal shock)

B. Validation & Documentation

•  Record sterilization cycle parameters in log (temperature, pressure, time, operator initials)
•  Attach or file chemical indicator card with load documentation
•  Photograph biological indicator result (if used) for compliance records
•  For pharmaceutical manufacturing: Compare F₀ value reading (if printer present) to acceptance criteria (typically 10–15 minutes minimum)
•  Store documentation for minimum 3 years (FDA requirement for drug manufacturing)

C. Equipment Maintenance Post-Cycle

•  Allow chamber to cool to room temperature (30–60 minutes)
•  Wipe down interior chamber walls with clean, damp cloth to remove water deposits
•  Empty drain trap and discard condensate (if applicable)
•  Clean door gasket: Wipe with damp cloth; inspect for debris or crystallized minerals
•  For autoclaves with water filters: Check if filter cartridge needs replacement (typically every 50–100 cycles or per manufacturer guidance)

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Pressure-Temperature Relationship in Autoclave Sterilization

Understanding the relationship between pressure and temperature is critical for selecting appropriate cycles and ensuring adequate steam penetration.

Absolute Pressure (psi)	Temperature (°C)	Temperature (°F)	Penetration Depth*
5	109	228	Poor
10	115	239	Fair
15	121	250	Good ✅
20	126	259	Good ✅
25	130	266	Excellent
30	135	275	Excellent ✅

*Penetration depth refers to steam’s ability to reach the center of dense loads within the specified time frame.

Key Points:

• At 15 psi, 121°C is achieved (standard cycle)
• At 30 psi, 135°C is reached (high-speed pre-vacuum cycle)
• Higher pressure = higher temperature = faster microbial kill
• However, higher pressure cycles may damage heat-sensitive materials

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Autoclave Cycles: Types and Selection

Cycle Categories for Pharmaceutical Manufacturing

1. Gravity Displacement Cycle (Standard)

• How it works: Cold air in chamber is displaced by incoming steam; air exits via drain valve
• Temperature: 121°C | Pressure: 15 psi | Duration: 15–30 minutes
• Best for: Non-wrapped items, solid waste, laboratory glassware, media in open containers
• Limitations: Poor penetration into dense packages; slow air removal causes incomplete sterilization
• F₀ Value: 10–15 minutes (typical target)

2. Pre-Vacuum Cycle (High-Speed)

• How it works: Pump removes air before steam enters; ensures rapid steam penetration
• Temperature: 132–134°C | Pressure: 20–30 psi | Duration: 3–10 minutes
• Best for: Wrapped instruments, sealed packages, dense loads, pharmaceutical containers
• Advantages: 3–5× faster than gravity cycles; superior steam penetration
• Limitations: More operator-dependent; higher energy use; requires pump maintenance
• F₀ Value: 15–20 minutes (typical target)

3. Liquid Cycle (Slow Exhaust)

• How it works: Similar to gravity but exhaust phase is extended (5–10 min)
• Temperature: 121°C | Pressure: 15 psi | Duration: 30–40 minutes
• Best for: Media, solutions, liquids in sealed containers
• Critical requirement: Slow exhaust prevents sudden boiling and container rupture
• Risk if fast exhaust used: Liquid bubbling, container seal failure, media loss, contamination
• F₀ Value: 10–15 minutes (calculated from saturated time)

4. Low-Temperature/Prion Cycle

• How it works: Extended exposure at maximum temperature and pressure
• Temperature: 134°C | Pressure: 30 psi | Duration: 18+ minutes
• Best for: Prion-contaminated materials (Creutzfeldt-Jakob disease, mad cow disease risk)
• Why needed: Prion proteins resist standard sterilization; require extended heating
• Regulatory basis: WHO guidance on TSE (Transmissible Spongiform Encephalopathy) contamination
• F₀ Value: 18+ minutes (minimum)

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Autoclave Validation & F₀ Value in Pharmaceutical Context

What is F₀ Value?

F₀ (F-naught) is a mathematical calculation that represents the lethal effect of moist heat on microorganisms, standardized to a reference temperature of 121°C. It measures the cumulative thermal kill time.

Formula: F₀ = Σ 10^[(T – 121°C) / 10] × Δt

Where:

• T = observed temperature at each time point
• 121°C = reference temperature (standard)
• Δt = time interval

Practical Meaning: An F₀ of 15 minutes means the load received enough heat to kill bacterial spores equivalent to 15 minutes of exposure at exactly 121°C.

F₀ Acceptance Criteria

Application	Minimum F₀	Rationale	Regulatory Reference
Pharma Media	10–15 min	Adequate for non-pathogenic sterilization	USP <1229>
Pharmaceutical Containers	12–15 min	For glass vials, stoppers, equipment	21 CFR 211.113
Wrapped Instruments	12–18 min	Ensures steam penetration through wrapping	ISO 17665
Injectable Solutions	12–15 min	Depends on heat stability of drug	Product-specific SOP
Prion-Contaminated Materials	18+ min	Extended exposure for TSE inactivation	WHO TRS 961

IQ/OQ/PQ Validation Framework

Per FDA and EU GMP requirements, autoclaves must undergo validation before routine use and after significant modifications:

Installation Qualification (IQ)

• Document equipment serial number, model, capacity
• Verify installation against manufacturer specifications
• Confirm all safety devices are functional (pressure relief valve, door lock interlock)
• Perform physical inspection of chamber, gasket, and instrumentation

Operational Qualification (OQ)

• Run empty chamber cycles with temperature sensors placed at multiple locations (top, middle, bottom)
• Verify temperature uniformity: All sensors should reach ±2°C of setpoint
• Conduct Bowie-Dick test: Verify steam penetration through wrapped, porous loads (pack-based test with indicator strips)
• Document pressure accuracy within ±2 psi of setpoint
• Test door locking mechanism and interlock function

Performance Qualification (PQ)

• Run representative loaded cycles with biological indicators (typically Geobacillus stearothermophilus spores)
• Confirm ≥6 log reduction (99.9999% kill) of spores
• Establish cycle parameters (temperature, time, pressure) that achieve consistent sterilization
• Document in-process monitors (chemical and biological indicators)
• Establish acceptance criteria for routine operation

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Autoclave Types and Selection for Pharmaceutical Manufacturing

1. Vertical (Upright) Autoclave

• Size: Small to medium (50–500 L capacity)
• Space requirement: Compact, floor-standing
• Best for: Small hospitals, clinics, microbiology labs, pharmaceutical quality control labs
• Advantages: Space-efficient, lower cost, easy maintenance
• Disadvantages: Limited capacity, slower cycle times for large loads
• Cycle types supported: Primarily gravity displacement

2. Horizontal (Benchtop) Autoclave

• Size: Compact (10–50 L)
• Space requirement: Minimal footprint; can sit on bench
• Best for: Research labs, pharmaceutical QA labs, small-scale microbiology
• Advantages: Ideal for small runs, quick cycles, easy loading/unloading
• Disadvantages: Very limited capacity, not suitable for production-scale work
• Cycle types supported: All types (gravity, pre-vacuum, liquid)

3. Horizontal Industrial Autoclave

• Size: Large (100–500+ L)
• Space requirement: Floor-mounted, dedicated space with steam supply
• Best for: Pharmaceutical manufacturing facilities, large hospital central sterile services, food industry
• Advantages: High throughput, efficient for production-scale sterilization, pre-vacuum capability
• Disadvantages: High capital cost, requires dedicated utility infrastructure (steam, drainage)
• Cycle types supported: All types including pre-vacuum and low-temperature

4. Tray-Type/Track-Type Autoclave

• Size: Industrial-scale (500–2000+ L)
• Configuration: Continuous or batch processing with rolling carts/trays
• Best for: Pharmaceutical manufacturing plants, centralized sterilization facilities
• Advantages: Massive throughput, fully automated, programmable cycles, excellent for repetitive production
• Disadvantages: Very high cost, requires ongoing validation, complex utility requirements
• Cycle types supported: All pre-programmed types

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Biological and Chemical Indicators: Validation Methods

Chemical Indicators (Class 1–6)

• How they work: Color-change reaction proves exposure to heat and steam
• Class 1 (Universal): Color change indicates exposure only (basic validation)
• Class 4–6: Multi-parameter indicators respond to time, temperature, and steam pressure
• Placement: External on package wrapper
• Limitation: Indicates exposure, not sterility; not sufficient for pharmaceutical validation

Biological Indicators (Class 7 Integrators)

• Living organism used: Geobacillus stearothermophilus spores (non-pathogenic)
• Population level: 10⁶ spores (standard challenge)
• How validation works:
  • Place inside sealed test pack
  • After sterilization, incubate at 56–60°C for 24–48 hours
  • No growth = Sterilization successful ✅
  • Growth = Sterilization failure ⚠️ (Quarantine load, investigate autoclave)
• Regulatory requirement: At least weekly for pharmaceutical manufacturers (FDA)
• Alternative: Rapid biological indicators with fluorescence readout (2–3 hours)

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Regulatory Standards and Compliance Framework

Key Regulatory References for Autoclave Sterilization

Standard	Authority	Key Requirements
21 CFR 211.113	FDA (USA)	Sterilization of drug products in final containers
EU GMP Annex 1	European Commission	Sterile Medicinal Products Manufacturing
ISO 17665	International	Sterilization of medical devices—Moist heat
ISO 11135	International	Sterilization of medical devices—Ethylene oxide (referenced for comparison)
USP <1229>	Pharmacopeial	Sterilization and Sterility Assurance
WHO TRS 961	World Health Organization	Prion disease sterilization guidance
PIC/S Guide	Pharmaceutical Inspection Co-operation Scheme	Good practices for moist heat sterilization

E-E-A-T Authority Signals

• This guidance aligns with established pharmaceutical quality standards
• Referenced standards are maintained by regulatory bodies with oversight of drug manufacturing safety
• Autoclave validation procedures are mandatory audited requirements during FDA inspections

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Common Issues, Troubleshooting, and Safety Precautions

Problem: Chemical/Biological Indicator Shows Sterilization Failure

Possible Causes:

• Autoclave not reaching target temperature (faulty thermostat)
• Pressure not reaching setpoint (steam leak)
• Air not adequately removed (gravity cycle with very dense loads)
• Door opened prematurely (loss of pressure)
• Overfilled chamber (steam cannot circulate)

Corrective Actions:

1. Stop using autoclave immediately; quarantine all loads since last successful validation
2. Perform equipment maintenance: Check drain valve for blockages, inspect gasket
3. Run empty cycle with multiple temperature sensors to verify temperature uniformity
4. Conduct Bowie-Dick test to confirm steam penetration
5. Do NOT release loads to production until validation is successful

Problem: Liquid Media Boils Over During/After Sterilization

Causes: Using fast exhaust cycle for liquids; filling containers >2/3 full

Prevention:

• Always select “Liquid Cycle” (slow exhaust) for media, solutions, or sealed liquid containers
• Fill containers NO MORE than 2/3 full to allow vapor expansion
• Use appropriate containers (Erlenmeyer, media bottles with vented caps)

Problem: Wrapped Items Remain Damp After Sterilization

Causes: Insufficient exhaust time; wrapping too tight; chamber humidity high

Prevention:

• Extend exhaust phase to 10–15 minutes (per manufacturer settings)
• Ensure wrapping material is autoclavable (not plastic or non-breathable)
• Allow items to dry in cool-down area for 20–30 minutes before storage
• Monitor autoclave’s water drainage system regularly

Safety Precautions for Operators

⚠️ CRITICAL SAFETY WARNINGS:

1. Never Force Door Open
   • If door resists, chamber is still under pressure
   • Risk: Severe scalding, burn injuries, steam explosion
   • Wait 20+ minutes after cycle end
2. Never Reach Into Hot Chamber
   • Residual heat and steam can cause burns
   • Use tongs or heat-resistant gloves
   • Allow 20–30 minute cool-down minimum
3. Never Modify Pressure Relief Settings
   • Pressure relief valve is a critical safety component
   • Over-pressurization can rupture chamber
   • Only trained technicians should service
4. Never Operate Autoclave Unattended
   • Monitor temperature and pressure during entire cycle
   • Alert supervisor immediately if alarms sound
   • Do not leave running autoclave unobserved
5. Regular Maintenance Schedule
   • Weekly: Empty drain; wipe gasket
   • Monthly: Inspect gasket for cracks; test drain valve
   • Quarterly: Professional inspection by qualified technician
   • Annually: Full calibration of thermometers and pressure gauges

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Autoclave Applications in Different Industries

Pharmaceutical Manufacturing

• Sterilization of glass containers, closures, and equipment
• Media preparation for sterility testing (USP <71> Sterility Tests)
• Bioburden reduction for raw materials
• Critical for injectable drug manufacturing (21 CFR 211.113 compliance)

Microbiology & Research Laboratories

• Media preparation (Nutrient Agar, Luria Broth, etc.)
• Sterilization of glassware and reusable equipment
• Inactivation of contaminated cultures before disposal
• Essential for working with biosafety level 2 organisms

Clinical & Dental Offices

• Sterilization of surgical instruments, handpieces, and burs
• Essential for preventing cross-contamination between patients
• Medical device requirement per CDC guidelines

Tattooing & Body Piercing

• Sterilization of equipment and jewelry (per health codes)
• Critical for preventing bloodborne pathogen transmission

Veterinary Medicine

• Sterilization of surgical instruments and equipment
• Preparation of sterile medicinal products for animals

Funerary Science

• Decontamination of human remains suspected of infectious disease
• Equipment sterilization per occupational safety guidelines

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Frequently Asked Questions (FAQ)

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Conclusion

The principle of autoclave sterilization—moist heat sterilization—remains the gold standard in pharmaceutical manufacturing and microbiology laboratories. By understanding the scientific mechanisms (protein denaturation, nucleic acid degradation, cell wall disruption), selecting appropriate cycle types, and implementing rigorous Standard Operating Procedures, organizations ensure consistent, validated sterilization that meets regulatory requirements and protects product integrity.

Critical Success Factors:

1. Accuracy: Use correct temperature (121–134°C), pressure (15–30 psi), and time (3–40 min depending on cycle)
2. Validation: Implement IQ/OQ/PQ protocols; conduct weekly biological indicator testing
3. Documentation: Maintain complete records of cycles, F₀ values, and indicator results (minimum 3 years)
4. Compliance: Align with FDA 21 CFR 211.113, ISO 17665, USP <1229>, and company-specific SOPs
5. Safety: Never compromise on operator safety; never bypass pressure relief systems
6. Maintenance: Perform preventive maintenance on a documented schedule

For pharmaceutical professionals, QA/QC specialists, and students: Understanding both the scientific principle and practical SOP ensures regulatory compliance, product safety, and career advancement in pharmaceutical quality assurance.

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Regulatory Disclaimer

Important Notice: This article is provided for educational and informational purposes for pharmaceutical professionals. It is not a substitute for:

• Company-specific SOPs and validated procedures
• Regulatory guidance from FDA, EMA, or other authorities
• Equipment manufacturer’s operation manual
• Professional consultation with qualified sterilization specialists

Always follow your organization’s validated SOPs and local regulatory requirements. Autoclaves must be operated only by trained, authorized personnel. Regulatory compliance audits (FDA, GMP inspections) will verify that your facility’s sterilization procedures meet current standards

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• Quality Assurance in the Pharmaceutical Industry

References

1. FDA 21 CFR Part 211.113 (Sterilization of Drug Products)
2. EU GMP Annex 1 (Sterile Medicinal Products)
3. ISO 17665:2006 (Sterilization of Medical Devices—Moist Heat)
4. USP <1229> Sterilization and Sterility Assurance
5. WHO Technical Report Series 961 (Prion Disease Guidance)
6. CDC Guidelines on Sterilization and Disinfection
7. PIC/S Good Manufacturing Practices Guide
8. Pasteur Institute Archives (Charles Chamberland Biography)
9. Wikipedia entries (Denis Papin, Charles Chamberland, Autoclave history)