Clean-in-place (CIP) is a widely used method of automated cleaning that is performed directly within manufacturing equipment with little or no equipment disassembly. It typically consists of a programmable
cleaning cycle with multiple steps. In each step, a specific cleaning fluid (that may be a solvent such as water or a solution of various cleaning chemicals) is continuously circulated through the system at a controlled flow rate, temperature and cleaning agent concentration, and for a set duration. CIP is widely used in the food, dairy, beverage, brewing, pharmaceutical, and cosmetic industries to clean large fixed equipment, such as fermenters, bioreactors, centrifuges, heat exchangers, tanks, and piping. Vessels and other large equipment are cleaned by spraying the cleaning fluid on product contact surfaces, thereby creating a falling film of fluid on the surface. Piping and associated components, such as valves and pumps, are cleaned by flooding them with the cleaning fluid. Advantages of CIP systems include low labor requirement, fast equipment turnaround, and consistency. High initial investment and equipment maintenance costs and relatively high usage of water, chemicals, and energy are the main drawbacks.

Small vessels and parts that can be readily disassembled are typically cleaned out of place in a parts washer or a bath with a continuous circulation loop. Clean-out-of-place (COP) processes can be manual, semiautomated, or fully automated. COP systems are generally more customizable, require less initial investment and maintenance, and consume less water, chemicals, and energy than CIP systems. Labor requirements and equipment turnaround times, however, are generally higher for COP processes, especially for manual and semiautomated systems. Custom designed parts washers are generally more effective and efficient at removing tenacious deposits in localized “hot spots” that are deemed to be difficult to clean. This is because the nozzles in a parts washer can provide high fluid impingement and shear at specific locations. Examples of tenacious deposits are cell debris and denatured proteins that dry on heated surfaces in heat exchangers and autoclaves. Surfaces that are difficult to access, such as the insides of pumps, valves, and filling needles, can be cleaned effectively with sonication, typically at frequencies between 18 and 120 kHz. COP baths are also used to pre-soak and pre-clean parts to reduce cycle-time or facilitate the use of milder cleaning conditions for manual cleaning, such as a lower temperature and cleaning agent concentration, and the use of milder cleaning agents such as a weak alkali or acid.

Analytical methods that are widely used to quantitate chemical contaminants include conductivity, total organic carbon (TOC), chromatography, and ultraviolet and fluorescence spectroscopy. Additionally, sodium dodecyl sulfate polyacrylamide gel electrophoresis is used to assess fragmentation and aggregation of protein during cleaning.⁵

Conductivity is mainly used to verify clearance of ionic contaminants and cleaning chemicals such as alkali and acid cleaners. TOC is widely used to quantitate organic contaminants. It is also a useful method for detecting biological residues on reusable medical devices (see chapter 47).⁶,⁷,⁸ The limit of quantitation (LOQ) of TOC is approximately 0.1 ppm, which is equivalent to approximately 5 µg/cm² for swab samples (R. Sharnez, unpublished data, 2019). In comparison, the visible residue limit of most process residues is between 1 and 4 µg/cm².⁹ A drawback of TOC is that samples can become contaminated with organic solvents and disinfecting agents, such as alcohol, which in turn can result in apparent failures and complex investigations. Also, being a nonspecific method, TOC measures organic carbon in the entire residue, not just in the contaminant. Thus, all of the measured carbon in the residue is attributed to the contaminant, a worst-case assumption that can greatly overestimate the concentration of the contaminant. Another drawback of TOC is that the recovery is sometimes too low to accurately quantify the amount of contaminant in a sample.¹⁰ Also, to accurately determine the recovery of a contaminant, the theoretical (ie, actual, not measured) carbon fraction of the contaminant in the residue is required. This information is seldom available for complex soils, such as human, plant and animal-based materials, proprietary culture media, and biological waste. These drawbacks are especially significant when the contaminant is composed of a relatively small fraction of the overall carbon in the residue. Product-specific assays, such as enzyme-linked immunosorbent assay and other immunoassays, overcome some of the limitations of TOC but can require more time and expertise to develop. They also have greater specificity and sensitivity; for example, the LOQ of most immunoassays is approximately 1 ppb.

Acceptance limits, when applicable, for bioburden and endotoxin, and alert and action limits for swab and rinse samples are discussed later in this chapter.

The cleanability of a soil depends on several factors including composition, thickness of the deposit, surface defects and corrosion, affinity to the surface, and cohesive forces within the soil matrix; swelling, degradation, and denaturation of the soil during cleaning; and moisture content (dryness) and porosity (if the soil is sufficiently dry). Most soils become harder to clean as they lose moisture, but some soils can become easier to clean, and even slough off the surface, when they are sufficiently dry. Thus, the post-use hold time and drying rate can be critical in evaluating the cleanability of a soil. The drying rate is primarily determined by humidity, temperature, and circulation rate of the surrounding air. Because of their strong adhesive bond with most materials, proteins are likely to be the first soil constituent to attach to equipment surfaces, forming the framework for other constituents to attach. The adhesive bonds between proteins and some surfaces can be quite strong, especially if the deposit is formed on a hot surface as in a heat exchanger. Also, denaturation and aggregation of proteins increases with temperature and pH, which generally makes them harder to remove.

Most cleaning processes remove surface residues by mechanical means (fluid shear or scrubbing) and chemical diffusion (Figure 8.1). These mechanisms are facilitated by wetting, chemical reactions (hydrolysis), and solubilization. Cleaning processes for biological residues generally rely on both shear and diffusion to adequately remove them.

The purpose of a pre-rinse is to wet the soil and physically remove it from the equipment. Wetting weakens the cohesive bonds between soil components, thereby facilitating their removal by fluid shear. It also opens up pathways for soil components to diffuse into the cleaning fluid, and for cleaning chemicals to diffuse into the soil matrix during subsequent chemical washes. As discussed earlier, because of the inverse relation between temperature and fluid shear, pre-rinses are generally more effective at a lower temperature. Pre-rinsing at a lower temperature also minimizes denaturation, aggregation, and subsequent redeposition of proteins and other biological macromolecules on equipment surfaces.

At sufficiently high fluid shear (typically of the order of 1 N/m²), the pre-rinse can remove over 90% of the soil. Under these conditions, the optimum duration of the pre-rinse is generally between 1 and 5 minutes. A more precise estimate of the optimum duration of the pre-rinse can be obtained by monitoring the concentration of solids in the effluent rinse water by measuring optical density, for example, and noting the time it takes for the effluent to become clear.

Alkaline cleaning chemicals such as sodium hydroxide (NaOH) can be very effective at removing proteins and other biological resides from device and equipment surfaces. Alkaline cleaning agents hydrolyze proteins into peptides and other fragments that are more soluble and therefore less likely to aggregate and redeposit on equipment surfaces. They can also enhance wetting and solubilization. These mechanisms are generally favored at higher NaOH concentration, that is, higher pH; however, most proteins denature and aggregate rapidly at NaOH concentrations above 1% and temperatures greater than 70°C (R. Sharnez, unpublished data, 2019). Because denatured protein aggregates are generally more difficult to clean, the optimum concentration and temperature of NaOH for cleaning proteinaceous soils is typically between 0.5% and 1% (pH approximately 12.5) and between 50°C and 70°C. Under optimum conditions, an alkaline wash can reduce most proteinaceous residues to visually undetectable levels within 5 to 10 minutes.

In some instances, the duration of the alkaline wash can be shortened or otherwise optimized by adding surfactants and wetting agents (as in formulated cleaning agents; see chapter 6). Nonionic wetting agents are good emulsifiers and can reduce foaming. Sequestrants, such as sodium phosphate derivatives, may be required to chelate minerals in hard water and prevent them from precipitating during the cleaning process. Chlorine compounds and peroxides, such as sodium hypochlorite (bleach) and hydrogen peroxide, can also enhance the effectiveness of alkaline cleaning agents; however, they can corrode stainless steel and other metals, especially at concentrations and temperatures above 1% and 50°C (R. Sharnez, unpublished data, 2019).

The alkaline wash is typically followed by an intermediate rinse to remove the cleaning agent and entrained particles.

An acid wash is sometimes necessary to remove inorganic constituents and precipitated minerals. It is also used to passivate stainless steel and other metal surfaces.

The purpose of the final rinse is to reduce residual cleaning chemicals and entrained particles to acceptable levels. The final rinse is performed with high purity water, such as water for injection (WFI), or distilled or deionized water.

The effectiveness of the earlier steps is determined by several equipment parameters such as contact between cleaning fluids and soiled surfaces (spray coverage), drainability of equipment surfaces, and effective control of critical process parameters (CPPs) such as flow rate and temperature and concentration of the cleaning fluids.¹⁴,¹⁵,¹⁶ Spray coverage is evaluated at full scale with a dilute aqueous suspension of riboflavin. Bench-scale cleanability studies are used to optimize CPPs¹²,¹⁷,¹⁸,¹⁹ and identify suitable master soils for developing and validating cleaning procedures at full scale.²⁰,²¹

Various other cleaning formulations and processes can be used depending on the type of soil and the acceptable level of the contaminant. These can be as simple as using high purity water alone, particularly for residues that are highly soluble in water, but can also include the following: