EP4731179A1

EP4731179A1 - Vaginal lubricant comprising acrylamide and cellulose compounds

Info

Publication number: EP4731179A1
Application number: EP24735962.3A
Authority: EP
Inventors: Siree PONGMANAWUT; Irene SEE; Adam Terry Simpson
Original assignee: Reckitt Benckiser Health Ltd
Current assignee: Reckitt Benckiser Health Ltd
Priority date: 2023-06-21
Filing date: 2024-06-20
Publication date: 2026-04-29
Also published as: CN121358464A; GB2631253B; AU2024311319A1; GB2631253A; WO2024261164A1

Abstract

An aqueous lubricant which has a pH of 4.5 or less, and an osmolality of 500 mOsm/kg or less, wherein the lubricant comprises: a copolymer comprising acrylamide and a monomer having a carboxylic acid or carboxylate group; and a thickener which is an alkylcellulose, hydroxyalkylcellulose, or carboxyalkylcellulose. The present invention also provides a product containing this lubricant and a method of manufacturing it. Use of said aqueous lubricant to provide lubrication and/or moisturisation to the skin or a mucous membrane of a subject, preferably to the vagina and/or during sexual activity. A method of manufacturing said aqueous lubricant, comprising dispersal of the thickener in water and, in a later step, addition of the copolymer using mixing at less than 1000 rpm; preferably comprising dispersal of the thickener in water with mixing at 1000 rpm or more.

Description

VAGINAL LUBRICANT COMPRISING ACRYLAMIDE AND CELLULOSE COMPOUNDS

DESCRIPTION

Technical field

The present invention is in the field of personal lubricants. In particular, it relates to a lubricant, a product comprising the lubricant, a use of the lubricant, and a method of manufacturing it.

Background

For a large proportion of each month, women of reproductive age can be naturally drier or at least not optimally lubricated in the vaginal region, due to hormonal changes that accompany their menstrual cycle. Many other factors can also influence natural lubrication levels across ages, including stress or anxiety, pregnancy, breastfeeding, hormonal contraceptives, diabetes, some cancer treatments, and douching practices. Vaginal dryness or sub-optimal lubrication may lead to discomfort during sex and ultimately a disinclination to engage in sex.

Personal lubricants are specialised lubricants which serve to reduce friction with body tissues during sexual activity. They may also be described as personal moisturisers, as they can be used at other times to reduce discomfort associated with vaginal dryness. The technical considerations for standalone lubricants / moisturisers tend to both differ from, for example, the type of lubricant which is pre-provided on the surface of a condom. The latter lubricants must be stable with the condom material and its packaging for storage periods of several years. This invention is focussed primarily on standalone products designed to be applied directly to the vagina, whether to enhance sexual activity or for additional lubrication or moisturisation outside of sexual activity. The consumer may of course still choose to apply the product to a penis, other intimate area, a condom, or sex toy.

A wide variety of standalone personal lubricants are currently available. They can be water-based or water-free (typically silicone-based), depending on the intended area of application and the required condom or toy compatibility. Silicone-based lubricants can damage sex toys made of silicone and may stain fabric, but tend to be longer-lasting; water-based personal lubricants are the most widely available type on the market globally.

Historically, water-based personal lubricants have tended to have an osmolality much higher than the typical vaginal osmolality in the range of 200-300 mOsm/kg. It is now appreciated that the osmolality of a lubricant is important, because the epithelial cells lining the vagina wall will constantly try and maintain homeostasis. Hyperosmolar lubricants cause the cells of the vagina to release fluid to dilute the lubricant. This may result in epithelial cell damage, which in turn results in an increased risk of infection due to the compromised epithelial barrier. There may also be increased dryness of the vagina. This is particularly problematic for menopausal females, as the use of the hyperosmolar lubricant exacerbates vaginal dryness.

On the other hand, hypo-osmolar lubricants (which are quite rare on the market) cause the vaginal cells to draw water from the lubricant. In the extreme, this could lead to swelling and ultimately rupture of the cells. The ideal lubricant would be iso-osmolar. However, formulating a personal lubricant within this range is not straightforward whilst also balancing the need to achieve a number of other parameters, including the required viscosity, slip properties, and rheological stability during storage. In 2012, the World Health Organisation (WHO) in its Advisory note WHO/UNFPA/FHI360 recommended that healthcare organisations procure personal lubricants which have an osmolality of less than 1200 mOsm/kg.

It is also now recognised that the pH of a water-based lubricant is important to control. A healthy vagina normally has a pH in the range 3.8 - 4.5; high pH can increase the risk of bacterial vaginosis. The WHO's Advisory note stated that ideally a vaginal lubricant should have a pH of about 4.5, but procurement agencies should purchase lubricants with a pH of 7 or less. This advice took into consideration the possibility of cross over use where a vaginal lubricant might be used rectally, the ideal rectal lubricant having a pH of about 5.5 to 7.

WO2022/153066 discloses lubricants with reduced hyperosmolality and relatively low pH. In recent times, a few vaginal lubricants have also started to appear on the market which have a pH and osmolality close to the natural environment of the vagina. Nevertheless, these do not have a physical texture that mimics natural vaginal fluid. Consumers may describe them as watery and/or feeling unnatural.

US3,965,908 disclosed many years ago a "synthetic physiological mucus" useful as a vaginal lubricant, with physical characteristics similar to natural mucus. It was based on a pituitous aqueous solution of a high molecular weight anionic polyacrylamide, preferably having 26-36% of the amide groups replaced by carboxyl groups. Although the polymer was said to be stable in the pH range of about 3 - 10, this document teaches that the desired mucus-like properties are only capable of being retained in the pH range of about 5 - 8. EP0014238A2 has a similar disclosure; although it states that the pH can be varied in the range 3.0 - 10.0 without changing the quality and physiological properties, it suggests that for the vaginal area a pH of about 5.0 should be used. The specific example formulations in these documents contain only the polyacrylamide, preservative(s), citric acid and water; though the osmolality is not mentioned, they are predicted to be strongly hypo-osmolar.

It would be advantageous to formulate a water-based vaginal lubricant which has a texture similar to natural vaginal fluid, whilst still being pH friendly and closer to iso- osmolar to the vagina.

Summary of the invention

In a first aspect of the invention there is provided a lubricant as set out in claim 1.

In a second aspect of the invention there is provided a product comprising the lubricant according to the invention in its first aspect that is provided inside a bottle or sachet, and packaged separately from any condom present in the product.

In a third aspect of the invention there is provided the use of the lubricant according to the invention in its first aspect to provide lubrication and/or moisturisation to the skin or a mucous membrane of a subject.

In a fourth aspect of the invention there is provided a method of manufacturing the lubricant according to the invention in its first aspect, comprising dispersal of the thickener in water and, in a later step, addition of the copolymer using mixing at less than 1000 rpm. Detailed description

Embodiments described herein in relation to one aspect of the invention may be combined with each other and may apply equally to other aspects of the invention, unless specified to the contrary or the context otherwise requires.

The lubricant of the invention comprises a copolymer of acrylamide and a monomer having a carboxylic acid or carboxylate group. Preferably, the copolymer is: a copolymer of acrylamide and a monomer having an acrylic acid group, preferably a copolymer of acrylamide and (alkyl)acrylic acid, preferably a copolymer of acrylamide and (C1-3 a I kyl)acry lie acid, preferably a copolymer of acrylamide and acrylic acid / methacrylic acid, preferably a copolymer of acrylamide and acrylic acid; or a salt of any of the foregoing. The copolymer may contain more than two monomers, but preferably the additional monomer(s) are also (alkyl)acrylic acid(s) or salt(s) thereof, for example the copolymer may be a terpolymer of acrylamide, acrylate and methacrylate (or the acid forms thereof). Preferably it is a copolymer of only two monomers. In an embodiment, the copolymer comprises a molar % of acrylamide groups of: at least 10 %, at least 15 %, at least 20 %, at least 25 %, at least 30 %, at least 35 %, at least 40 %, at least 45 %, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, or at least 75%; and/or up to 90 %, up to 85 %, up to 80 %, up to 75 %, up to 70 %, up to 65 %, up to 60 %, up to 55 %, or up to 50 %. In an embodiment, the copolymer comprises a molar % of acrylamide groups of 10 - 90 %, 40 - 85 %, or 60 - 75 %.

Preferably, the copolymer is provided in salt form, preferably an alkali metal salt, preferably as the sodium or potassium salt, preferably as the sodium salt. For example, the copolymer may be a copolymer of acrylamide and sodium acrylate. Preferably the copolymer used to make the lubricant is comprised in a raw material comprising very low residual content of acrylamide monomer, preferably no more than 0.05 wt%, no more than 0.04 wt%, no more than 0.03 wt%, or no more than 0.02 wt%.

In an embodiment, the copolymer has a weight average molecular weight of: at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da, or at least 5,000,000 Da; and/or up to 10,000,000 Da, up to 9,000,000 Da, up to 8,000,000 Da, up to 7,000,000 Da, up to 6,000,000 Da, or up to 5,000,000 Da. In an embodiment, the copolymer has a weight average molecular weight of 1,000,000 - 10,000,000 Da, preferably 2,000,000 - 6,000,000 Da, preferably 3,000,000 - 5,000,000 Da.

Suitable polyacrylamide copolymers can be manufactured according to known synthetic procedures or obtained commercially, for example from the Praestol™ range from Solensis, such as Praestol LT27 AP AG.

Preferably, the lubricant comprises the copolymer in an amount of: at least 0.01 wt%, at least 0.02 wt%, at least 0.03 wt%, at least 0.04 wt%, at least 0.05 wt%, at least 0.06 wt%, at least 0.07 wt%, at least 0.08 wt%, at least 0.09 wt%, at least 0.1 wt%, at least 0.15 wt%, or at least 0.2 wt%; and/or up to 0.5 wt%, up to 0.45 wt%, up to 0.4 wt%, up to 0.35 wt%, up to 0.3 wt%, up to 0.25 wt%, up to 0.20 wt%, up to 0.15 wt%, or up to 0.10 wt%. In an embodiment, the lubricant comprises the copolymer in an amount of 0.01 - 0.5 wt%, 0.1 - 0.4 wt%, 0.15 - 0.25 wt%, 0.2 wt% or about 0.2 wt%. In an embodiment, the lubricant comprises the copolymer in an amount of 0.05 - 0.15 wt%, 0.06 - 0.10 wt%, 0.07 - 0.09 wt%, 0.08 wt% or about 0.08 wt%.

The present inventors have found that the polyacrylamide copolymer may not be as efficient a thickener in lower pH ranges, in the inventive lubricant. To increase the viscosity of the formulation whilst maintaining the stringy mucus-like quality, a carefully- chosen thickener is used in combination with the copolymer. The thickener is an alkylcellulose (such as methylcellulose), hydroxyalkylcellulose or carboxyalkylcellulose (such as carboxymethylcellulose). In an embodiment, the lubricant comprises more than one of these thickeners. In an embodiment, the lubricant comprises another, different thickener from these thickeners, but preferably it comprises no other thickener besides alkylcellulose, hydroxyalkylcellulose and/or carboxylalkylcellulose (barring the polyacrylamide copolymer which may itself contribute to thickening).

The thickener is preferably one that allows the production of a transparent gel with good flowing property. It should also be capable of thickening the lubricant within the target pH range of the lubricant. During development, the present inventors tested xanthan gum and this was found to adversely affect the transparency and did not give a smoothly-flowing product. Another known thickener, Aristoflex AVC, did not thicken well in the low pH environment.

In an embodiment, the thickener used in the invention is a hydroxyalkylcellulose such as hydroxymethylcellulose or hydroxyethylcellulose, preferably hydroxyethylcellulose.

Preferably, the lubricant comprises the thickener in an amount of: at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, or at least 0.6 wt%, at least 0.7 wt%, or at least 0.8 wt%; and/or up to 1.0 wt%, up to 0.9 wt%, up to 0.8 wt%, or up to 0.7 wt%.

In an embodiment, the lubricant comprises the thickener in an amount of 0.2 - 1.0 wt%, preferably 0.4 - 0.9 wt% or 0.4 - 0.8 wt%, preferably 0.6 - 0.9 wt%, 0.6 - 0.7 wt%, 0.7 - 0.8 wt%, about 0.6 wt% or about 0.8 wt%. Preferably, the total amount of thickener (excluding the amount of the copolymer where this itself exerts a thickening effect) is within the foregoing ranges.

In an embodiment, the lubricant comprises a humectant. Many polyol compounds act as humectants, especially glycols, and triols like glycerol. The use of glycerol is less preferred, however. There is some suggestion in the field that it can act as a food source for Candida albicans, the yeast involved in vaginal candidiasis. The WHO Advisory note suggests using a total glycol content below approx. 8.3 wt% in order to limit the osmolality. However, the present inventors have recognised that the particular choice of glycol can also influence the properties. Polymeric glycols such as polyalkylene glycols confer a lower osmolality, but may lead to a stickier feeling product. On the other hand, 1,3-propane diol may also bring a sticky texture.

In an embodiment, the lubricant comprises a monomeric glycol, preferably a glycol having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably a glycol having 3 - 5 carbon atoms, preferably 3 or 4 carbon atoms, preferably 3 carbon atoms. Preferably, the lubricant comprises propylene glycol (1,2-propane diol). In an embodiment, the lubricant is free or essentially free of glycerol and/or 1,3-propane diol and/or polymeric glycol. In an embodiment, it contains less than 1 wt%, less than 0.5 wt%, less than 0.1 wt%, less than 0.05 wt%, or less than 0.01 wt%, of any or each of glycerol, 1,3-propane diol or polymeric glycol.

Preferably, the lubricant comprises propylene glycol in an amount of: at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1.0 wt%, at least 1.1 wt%, at least 1.2 wt%, at least 1.3 wt%, at least 1.4 wt%, at least 1.5 wt%, at least 1.6 wt%, at least 1.7 wt%, at least 1.8 wt%, at least 1.9 wt%, or at least 2.0 wt%; and/or up to 4.0 wt%, up to 3.9 wt%, up to 3.8 wt%, up to 3.7 wt%, up to 3.6 wt%, up to

3.5 wt%, up to 3.4 wt%, up to 3.3 wt%, up to 3.2 wt%, up to 3.1 wt%, up to 3.0 wt%, up to 2.9 wt%, up to 2.8 wt%, up to 2.7 wt%, up to 2.6 wt%, up to 2.5 wt%, up to 2.4 wt%, up to 2.3 wt%, up to 2.2 wt%, up to 2.1 wt%, or up to 2.0 wt%. In an embodiment, the lubricant comprises propylene glycol in an amount of 0.5 - 4.0 wt%, preferably 1.0 - 3.0 wt%, preferably 1.5 - 2.5 wt%, preferably 2.0 wt% or about 2 wt%. In an embodiment, the total amount of glycol (or polyol) is within these same ranges. In an embodiment, the only polyol(s) present are glycol(s). In an embodiment, propylene glycol is the only polyol present.

In an embodiment, the pH of the inventive lubricant is: at least 3.0, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, or at least 3.8; and/or up to 4.5, up to 4.4, up to 4.3, up to 4.2, up to 4.1, up to 4.0, up to 3.9, or up to 3.8. In an embodiment, its pH is 3.0 - 4.5, 3.2 - 4.5, 3.5 - 4.5, 3.5 - 4.4, 3.8 - 4.3, 3.8 or about 3.8. In particular, these pH values may be measured at 25 °C.

The required pH can be achieved using any suitable pH adjusting agent in an appropriate amount. For example, an organic acid such as lactic acid or citric acid may be used. Lactic acid is preferred because it is naturally found in healthy vaginas as the metabolic product of lactobacilli species present there. Vaginal lactic acid has been shown to have benefits including enhancing epithelial cell function and immune modulation.

Preferably, the lubricant contains a pH buffer, for example an organic acid in combination with a corresponding salt. In an embodiment, this is provided by lactic acid in combination with a lactate salt, such as potassium lactate.

The osmolality of normal female vaginal secretions is 260 - 290 mOsm/kg. The inventive lubricant has an osmolality of 500 mOsm/kg or less, and preferably has an osmolality: up to 490 mOsm/kg, up to 480 mOsm/kg, up to 470 mOsm/kg, up to 460 mOsm/kg, up to 450 mOsm/kg, up to 440 mOsm/kg, up to 430 mOsm/kg, up to 420 mOsm/kg, up to 410 mOsm/kg, up to 400 mOsm/kg, up to 390 mOsm/kg, or up to 380 mOsm/kg; and/or at least 100 mOsm/kg, at least 110 mOsm/kg, at least 120 mOsm/kg, at least 130 mOsm/kg, at least 140 mOsm/kg, at least 150 mOsm/kg, at least 160 mOsm/kg, at least 170 mOsm/kg, at least 180 mOsm/kg, at least 190 mOsm/kg, at least 200 mOsm/kg, at least 210 mOsm/kg, at least 220 mOsm/kg, at least 230 mOsm/kg, at least 240 mOsm/kg, at least 250 mOsm/kg, or at least 260 mOsm/kg. In an embodiment, the lubricant has an osmolality of 100 - 500 mOsm/kg, 200 - 500 mOsm/kg, 200 - 400 mOsm/kg, 250 - 400 mOsm/kg, or 260 - 380 mOsm/kg.

Any suitable preservative(s) can be used in the formulation. Preferably, the formulation contains substantially no parabens. Preferably, it is paraben-free. Studies have raised possible health concerns surrounding parabens, for example in relation to effects on hormone regulation and breast cancer risk. The preservative used in the present invention may optionally be an acid with a pKa in the range of 2 - 5. A preferred preservative in the present invention is benzoic acid, which has a pKa of 4.2.

In an embodiment, the lubricant comprises water in an amount of at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, or at least 96 wt%.

Preferably, the lubricant is clear and/or colourless. However, if desired, suitable dyes or colourants could be added in suitable amounts as will be known to the skilled person.

Preferably, the lubricant has a viscosity (as measured by a Brookfield viscometer) of: at least 200 cPs, at least 250 cPs, at least 300 cPs, at least 350 cPs, at least 380 cPs, or at least 400 cPs; and/or up to 1500 cPs, up to 1400 cPs, up to 1300 cPs, up to 1200 cPs, up to 1100 cPs, up to 1000 cPs, up to 900 cPs, or up to 800 cPs. In an embodiment, the lubricant has a viscosity of 200 - 1500 cPs, 300 - 1000 cPs or 400 - 800 cPs. Preferably, these viscosities are measured by a Brookfield viscometer using a spindle RV3 at 50 rpm and 25°C.

Another aspect of the invention is a product comprising the inventive lubricant, provided inside a bottle or sachet, and packaged separately from any condom present in the product. The bottle may, for example, be a container with a simple nozzle, or pump dispenser. The product may be a kit comprising a lubricant sachet and a separately- packaged condom, for instance where the sachet contains a small sample of the lubricant for trial purposes.

Another aspect of the invention is the use of the inventive lubricant to provide lubrication and/or moisturisation to the skin or a mucous membrane of a subject. Preferably, the lubricant provides lubrication and/or moisturisation to the vagina or other intimate area. Preferably, the lubricant is used during sexual activity. The user may apply the lubricant directly to the vagina or other intimate area, or onto a condom. In an embodiment, the lubricant is used to relieve vaginal dryness.

The lubricant may be manufactured using standard techniques known to those in the art. Preferably, high shear mixing (more than 1000 rpm) is avoided once the polyacrylamide copolymer has been added, to maintain the desirable stringy texture. However, high shear mixing may be desirable before addition of the polyacrylamide copolymer to disperse other ingredients at lower temperatures, such as the thickener. For example, the manufacturing method may comprise dispersal of hydroxyethylcellulose in water using a high shear homogenizer, and subsequent addition of polyacrylamide copolymer with low shear mixing. Preferably the entire manufacturing process is conducted at a temperature of no more than 40 °C, no more than 35 °C or no more than 30 °C. Another aspect of the invention relates to a manufacturing method for the inventive lubricant. The inventive method may have the advantage of a shorter manufacturing time and/or lower energy consumption. Examples

The following non-limiting examples are intended to illustrate the present invention.

Example 1

* Praestol LT27 AP or Praestol LT27 AP AG

** Purac BF P/41 (contains lactic acid and potassium lactate)

The inventive formulations of these examples were prepared by dispersing the hydroxyethylcellulose in water with a homogenizer until homogeneous, followed by slow gentle addition of the polyacrylamide copolymer with an overhead stirrer until completely swelled and no lumps observed. The remaining ingredients were added with continued gentle stirring. No heating was applied during the process.

Claims

1. An aqueous lubricant which has a pH of 4.5 or less, and an osmolality of 500 mOsm/kg or less, wherein the lubricant comprises: a copolymer comprising acrylamide and a monomer having a carboxylic acid or carboxylate group; and a thickener which is an alkylcellulose, hydroxyalkylcellulose, or carboxyalkylcellulose.

2. An aqueous lubricant as claimed in claim 1, wherein the copolymer comprises acrylamide and an acrylate, preferably wherein the copolymer is a copolymer of acrylamide and sodium acrylate.

3. An aqueous lubricant as claimed in claim 1 or 2, wherein the thickener is a hydroxyalkylcellulose, preferably hydroxyethylcellulose.

4. An aqueous lubricant as claimed in any of the preceding claims, comprising the thickener in an amount of: at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, or at least 0.8 wt%; and/or up to 1.0 wt%, up to 0.9 wt%, up to 0.8 wt%, or up to 0.7 wt%.

5. An aqueous lubricant as claimed in any of the preceding claims, comprising the copolymer in an amount of: at least 0.01 wt%, at least 0.02 wt%, at least 0.03 wt%, at least 0.04 wt%, at least 0.05 wt%, at least 0.06 wt%, at least 0.07 wt%, at least 0.08 wt%, at least 0.09 wt%, at least 0.1 wt%, at least 0.15 wt%, or at least 0.2 wt%; and/or up to 0.5 wt%, up to 0.45 wt%, up to 0.4 wt%, up to 0.35 wt%, up to 0.3 wt%, up to 0.25 wt%, up to 0.20 wt%, up to 0.15 wt%, or up to 0.10 wt%.

6. An aqueous lubricant as claimed in any of the preceding claims, comprising a monomeric glycol, preferably a glycol having 3 - 5 carbon atoms, preferably propylene glycol.

7. An aqueous lubricant as claimed in any of the preceding claims, wherein the total amount of polyol in the lubricant is 0.5 - 4.0 wt%, preferably wherein propylene glycol is the only polyol in the lubricant.

8. An aqueous lubricant as claimed in any of the preceding claims, which has an osmolality in the range 200 - 500 mOsm/kg, preferably 250 - 400 mOsm/kg, preferably 260 - 380 mOsm/kg.

9. An aqueous lubricant as claimed in any of the preceding claims, which has a pH in the range 3.2 to 4.5, preferably 3.5 to 4.4, preferably 3.8 to 4.3.

10. An aqueous lubricant as claimed in any of the preceding claims, which has a viscosity as measured by Brookfield viscometer of: at least 200 cPs, at least 250 cPs, at least 300 cPs, at least 350 cPs, at least 380 cPs, or at least 400 cPs; and/or up to 1500 cPs, up to 1400 cPs, up to 1300 cPs, up to 1200 cPs, up to 1100 cPs, up to 1000 cPs, up to 900 cPs, or up to 800 cPs.

11. A product comprising an aqueous lubricant as claimed in any of the preceding claims that is provided inside a bottle or sachet, and packaged separately from any condom present in the product.

12. Use of an aqueous lubricant as claimed in any of claims 1 to 10 to provide lubrication and/or moisturisation to the skin or a mucous membrane of a subject, preferably to the vagina and/or during sexual activity.

13. A method of manufacturing an aqueous lubricant as claimed in any of claims 1 to 10, comprising dispersal of the thickener in water and, in a later step, addition of the copolymer using mixing at less than 1000 rpm; preferably comprising dispersal of the thickener in water with mixing at 1000 rpm or more.

14. A method as claimed in claim 13, wherein the temperature is maintained at no more than 40 °C throughout the method.