Cell lysing agent comprising polyol / monool - epoxy products, composition, preparation, and method of use thereof

Abstract

The present application provides a lysis agent for a cell comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group (or) (i) reactant B: a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000 Daltons. The application also provides a cell lysis composition and a method for lysing a cell. The present application further provides a process for inactivating an enveloped virus

Description

CELL LYSING AGENT COMPRISING POLYOL / MONOOL - EPOXY PRODUCTS, COMPOSITION, PREPARATION, AND METHOD OF USE THEREOFFIELD OF THE INVENTION

[0001] The present invention provides a lysing agent for a cell comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group. Preferably, the reaction product is of (i) reactant A: an aliphatic hydrophilic polyether polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group.BACKGROUND OF THE INVENTION

[0002] Cell lysis is a commonly practiced method for the recovery of biological products from within cells. In many cases, the cells are contacted with a lysis reagent, commonly a solution comprising a detergent, or a solution of a lysis enzyme. The biological product can then be recovered from the lysis solution.

[0003] US Patent Publication 20080188673A1 describes low surface tension surfactants based on ether alcohol and their use as surfactants in aqueous coating formulations, the surfactants being prepared by reacting with at least one hydroxy compound.

[0004] JP Patent Publication 2011088113 A describes an emulsifier that contains a compound which is obtained by reaction of (a) glycidyl ethers with (b) water-soluble polyalkylene glycol having hydroxyl groups at both terminals of a molecular chain to form an adduct and then by reaction of the adduct with (c) organic diisocyanate.

[0005] US Granted Patent 7319021B2 discloses a cell lysis composition, methods for extracting and isolating proteins and peptides from a host cell using compositions, kits and apparatus.

[0006] Surfactants are used in cell lysis solutions because they disrupt the distinct interface between hydrophobic and hydrophilic systems. They help to solubilize membrane proteins and lipids, thereby causing the cell to lyse and release its contents. Surfactants are comprised of a polar hydrophilic head group and a nonpolar hydrophobic tail. They are categorized by the nature of the head group as either ionic, nonionic or zwitterionic.

[0007] Surfactant-based lysis is a popular method for cell rupture. It is easy to do and does not require any special equipment. However, surfactant cell lysis is often too mild, and needs to be donein conjunction with a physical method like grinding or homogenization. Also, harsh detergents can often damage or destroy the contents of the cell if used incorrectly.

[0008] There is need for a surfactant cell lysing agent that gently ruptures cells without damaging the valuable intracellular materials and a process for the use thereof that doesn't use toxic chemicals and that is fully scalable to manufacturing.SUMMARY OF THE INVENTION

[0009] In one important aspect, the present application provides a lysing agent for a cell comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group; (or) (i) reactant B: a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2; (or) wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4:1 to 1: 1.

[0010] In another important aspect, the present application provides a lysing agent for a cell comprising a reaction product of: (i) reactant A: a polyethylene glycol; and (ii) reactant B: 2- ethylhexyl glycidyl ether; wherein the ratio of molar equivalents of hydroxyl groups in the polyethylene glycol to molar equivalents of epoxy groups in 2-ethylhexyl glycidyl ether is about 1 : 1; wherein the reaction product comprises a mixture of polyethylene glycol mono-functionalized by 2- ethylhexyl glycidyl ether, polyethylene glycol di-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tri-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tetrafunctionalized by 2-ethylhexyl glycidyl ether, and polyethylene glycol penta-functionalized by 2- ethylhexyl glycidyl ether; and wherein the reaction product has the following generic structure:wherein each C11H22O2 unit is independentlywherein each Ci 1H22O2 unit is attached to the polyethylene glycol core or another C11H22O2 unit by a carbon-oxygen single bond; wherein the sum of all n ’s is an integer ranging from 1 to about 90; m is an integer ranging from 1 to 7; a and b are integers, wherein a + (m x b) ranges from 1 to (5 x / ??); and Q is an (m + 1) valent alkylene group comprising two to twenty carbon atoms, optionally branched, optionally substituted, and optionally containing ether, oxa or aza moieties; and wherein, if Q = -CH2CH2- and m = 1, the reaction product comprises: polyethylene glycol monofunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 1; polyethylene glycol difunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 2; polyethylene glycol trifunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 3; polyethylene glycol tetrafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 4; and polyethylene glycol pentafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 5.

[0011] In another important aspect, the present application provides a cell lysis composition comprising: (a) from 0.01 wt.% to 0.5 wt.% of the lysing agent; (b) cells having a desired protein or peptide; and (c) water, buffer, or a combination thereof; wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof; selected from the group of bacterial, yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NS0 (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9, and S2 (Drosophila , and wherein the yeast cells are selected from the group consisting of S. cerevisiae, and P. pastoris.

[0012] In another important aspect, the present application provides a process for producing a biological product, comprising the steps of: (a) culturing host cells producing the biological product; (b) lysing the cells by the process comprising: (bl) providing a source of cells, (b2) adding a lysing agent and (b3) mixing at a temperature of 4 °C to 90 °C; and (c) recovering the biological product selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

[0013] In another aspect, the present application provides a process for inactivating an enveloped virus in a biological product comprising contacting the enveloped virus with an amount of at least 0.01 wt.% of lysing agent sufficient to inactivate the virus while recovering the biological product selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 illustrates the cell viability results of Example 10, using the reaction product of Example 4 (300).

[0015] Figure 2 illustrates the cells of Example 10 before treatment.

[0016] Figure 3 illustrates the cells of Example 10, after exposure to the reaction product of Example 4 (300).

[0017] Figure 4 illustrates untreated A549 cells of Example 11.

[0018] Figure 5 illustrates EGFP transfected A549 cells of Example 11 prior to treatment.

[0019] Figure 6 illustrates EGFP transfected A549 cells of Example 11 treated with 0.5% Triton X- 100 after 24hrs.

[0020] Figure 7 illustrates EGFP transfected A549 cells of Example 11 treated with 0.05% Triton X-100 after 24hrs.

[0021] Figure 8 illustrates EGFP transfected A549 cells of Example 11 treated with 0.01% Triton X-100 after 24hrs.

[0022] Figure 9 illustrates fluorescence intensity of EGFP of cells of Example 11 treated with Triton X -100.

[0023] Figure 10 illustrates EGFP transfected A549 cells of Example 11 treated with 0.5% of the reaction product of Example 7 (1000) after 24hrs.

[0024] Figure 11 illustrates EGFP transfected A549 cells of Example 11 treated with 0.05% of the reaction product of Example 7 (1000) after 24hrs.

[0025] Figure 12 illustrates EGFP transfected A549 cells of Example 11 treated with 0.01% of the reaction product of Example 7 (1000) after 24hrs.

[0026] Figure 13 illustrates fluorescence intensity of EGFP of cells of Example 11 treated with the reaction product of Example 7 (1000).

[0027] Figure 14 illustrates EGFP transfected A549 cells of Example 11 treated with 0.5% of the reaction product of Example 9 (3350) after 24hrs.

[0028] Figure 15 illustrates EGFP transfected A549 cells of Example 11 treated with 0.05% of the reaction product of Example 9 (3350) after 24hrs.

[0029] Figure 16 illustrates EGFP transfected A549 cells of Example 11 treated with 0.01% of the reaction product of Example 9 (3350) after 24hrs.

[0030] Figure 17 illustrates fluorescence intensity of EGFP of cells of Example 11 treated with the reaction product of Example 9 (3350).

[0031] Figure 18 illustrates fluorescence intensity of EGFP of cells of Example 11 comparing treatments with Triton X-100 and the reaction products of Examples 7 or 9 at 24hrs.

[0032] Figure 19 illustrates transfected A549 cells of Example 11 without treatment after 24hrs.

[0033] Figure 20 illustrates SF-9 Cells of Example 12 prior to exposure.

[0034] Figure 21 illustrates SF-9 Cells of Example 12 post exposure to Triton X-100.

[0035] Figure 22 illustrates SF-9 Cells of Example 12 post exposure to the reaction products of Example 7 (1000).

[0036] Figure 23 illustrates SF-9 Cells of Example 12 post exposure to the reaction product of Example 9 (3350).

[0037] Figure 24 illustrates Bovine red blood cell lysis of Example 13 after 1 hour incubation with Triton X-100 and the reaction products of Example 7 measured by absorbance of released hemoglobin

[0038] Figure 25 illustrates Stability of hemoglobin of Example 13 in the presence of Triton X-100 and the reaction products of Example 7.

[0039] Figure 26 illustrates Log Reduction of Retrovirus-Like-Particles of Example 13 after 60 Minutes Incubation with Triton X-100 and the reaction products of Example 7.

[0040] Figure 27 illustrates N-phenyl-l-napthylamine dye fluorescence versus the concentration of the reaction products of Example 7 with inflection point corresponding to critical micelle concentration.

[0041] Figure 28 illustrates Biodegradation of the reaction products of Example 7 in OECD301F test of Example 14.DETAILED DESCRIPTION OF THE INVENTION

[0042] The disclosed and claimed inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components and steps or methodologies set forth in the following description. The disclosed and claimed inventive concept(s) is capable of other aspects or of being practiced or carried out in various ways. The phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0043] Unless otherwise defined herein, technical terms used in connection with the disclosed and claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0044] The singular forms "a," "an," and "the" include plural forms unless otherwise specified or clearly implied to the contrary by the context. The terms “comprising” and “comprised of’ include the more restrictive meanings “consisting essentially of’ and “consisting of.”

[0045] All percentages, parts, proportions, and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.

[0046] All publications, articles, papers, patents, patent publications, and other references cited herein are incorporated herein in their entirety for all purposes to the extent consistent with the disclosure herein.

[0047] As used herein, the following terms have the meanings set out below.

[0048] As used herein, the use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more depending on the term to which it is attached.

[0049] As used herein, the term “aliphatic hydrophilic polyol” refers to a substance, i.e., reactant A, in one embodiment, which is a hydrophilic compound having two or more hydroxyl groups. Examples of aliphatic hydrophilic polyols include oligomers, polymers and copolymers of ethyleneoxide, propylene oxide, and butylene oxide, polyethylene glycols, polypropylene glycols, poly(trimethylene oxide) glycols, polytetramethylene ether glycols, polyurethane polyols, polycarbonate polyols, polyester polyols, and acrylic polyols.

[0050] As used herein, the term “aliphatic hydrophilic polyether polyol” refers to a substance, i.e., reactant A, in another embodiment, which is a polyether having two or more hydroxyl groups. Examples of aliphatic hydrophilic polyether polyols include oligomers, polymers and copolymers of ethylene oxide, propylene oxide, and butylene oxide, polyethylene glycols, polypropylene glycols, poly(trimethylene oxide) glycols and polytetramethylene ether glycols. In a preferred embodiment, the aliphatic hydrophilic polyether polyol is a polyethylene glycol.

[0051] As used herein, the term “aliphatic hydrophilic alkoxylated alcohol” refers to a substance, i.e., reactant C, having a structure set out below:wherein R is a linear or branched alkyl group having from 1 to about 20 carbons; n > 1; and, for each repeat unit, independently, R1= R2= H, R1= H and R2= CH3, or R1= CH3 and R2= H.

[0052] The term "cells" as used herein refers to biological cells, i.e. prokaryotic and eukaryotic cells.

[0053] The term "cell suspension" means a liquid that contains cells, and these cells can exist in a state grown in suspension or as adherent cells.

[0054] As used herein, the term "cell lysis" as used herein refers to a disruption of one or more cells such that the cells’ outer layer is permanently or temporarily disrupted to release their contents for further analysis, modification, or use.

[0055] As used herein, the term “cell lysis composition” refers to a composition that effects lysis or rupture of all or a portion of a cell population, tissues, or organisms used as the source of the protein and peptide molecules to be isolated, such that the soluble protein and peptide molecules (or portion thereof) that are contained in the cell, tissue, or organism source are released from the cell, tissue, or organism.

[0056] As used herein, the terms “cell lysis” or “cell lysis agent” or “cell lysing agent” or “cell lysing” relate to same concept of lysis, rupture of all or a portion of a cell population, tissues, or organisms and are used interchangeably throughout the specification.

[0057] As used herein, the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0058] As used herein, the term “functionalized” with reference to any moiety refers to the presence of one or more functional groups in the moiety. Various functional groups may be introduced in a moiety by way of one or more functionalization reactions known to a person having ordinary skill in the art. It is to be understood that an alcohol or polyol “functionalized” by an epoxy compound or glycidyl ether has reacted so that at least one hydroxyl group of the alcohol or polyol has formed an ether bond to one of the carbon atoms of an epoxide group of the epoxy compound or glycidyl ether so that at least one epoxide group of the epoxy compound or glycidyl ether has ring opened upon attack by the alcohol or polyol to generate a new hydroxyl group.

[0059] As used herein, the term “hydrophilic” refers to substances having a tendency to mix with, dissolve in, or be wetted by water. Hydrophilic substances have a strong affinity for water.

[0060] As used herein, the e term “hydrophobic” refers to substances having a tendency not to mix with, dissolve in, or be wetted by water. Hydrophobic substances do not have a strong affinity for water and tend to repel or fail to mix with water.

[0061] As used herein, the term “hydrophobic compound having an epoxy group” refers to a substance, i.e., reactant B, which is a hydrophobic substance having an epoxide group. An epoxy group is a functional group consisting of an oxygen atom joined by single bonds to two adjacent carbon atoms, thus forming the three-membered epoxide ring. The hydrophobic compound having an epoxy group may be an alkyl glycidyl ether in which the alkyl group is saturated or unsaturated, straight-chain or branched aliphatic hydrocarbyl group having from 3 to 24 carbon atoms. Preferably, the hydrophobic compound having an epoxy group is a primary alkyl glycidyl ether, such as n-butyl glycidyl ether, n-octyl glycidyl ether, n-decyl glycidyl ether, n-dodecyl glycidyl ether, n-tetradecyl glycidyl ether, n-hexadecyl glycidyl ether, n-octadecyl glycidyl ether, n-octadecenyl glycidyl ether (oleyl glycidyl ether), docosyl glycidyl ether, and the like; a primary branched alkyl glycidyl ether, such as 2-ethylhexyl glycidyl ether, 2-hexyldecyl glycidyl ether, 2- octyldodecyl glycidyl ether, 2-heptylundecyl glycidyl ether, 2-(l,3,3-trimethylbutyl)octyl glycidyl ether, 2-decyltetradecyl glycidyl ether, 2-dodecylhexadecyl glycidyl ether, 2-tetradecyloctadecyl glycidyl ether; a secondary alkyl glycidyl ether such as sec-decyl glycidyl ether, sec-octyl glycidyl ether, sec-dodecyl glycidyl ether, and the like; or a tertiary alkyl glycidyl ether, such as t-octyl glycidyl ether, t-dodecyl glycidyl ether, and the like.

[0062] As used herein, the term “moiety” or “moieties” refers to a part(s) or a functional group(s) of a molecule.

[0063] As used herein, the term “polyethylene glycol” refers to both linear polyethylene glycols and branched polyethylene glycols, which may be formed by ethoxylating a polyol having three or more hydroxyl groups. Polyols having three or more hydroxyl groups include but are not limited to glycerol, trimethylolmethane, trimethylolethane, trimethylolpropane, benzenetriol, triethanolamine, threitol, erythritol, pentaerythritol, diglycerol, bis(trimethylolpropane), ribitol, xylitol, arabinitol, triglycerol, sorbitol, dipentaerythritol, tetraglycerol, pentaglycerol and hexaglycerol.

[0064] As used herein, the term “protein functionality” refers to properties of a protein molecule used for industrial or therapeutic applications such as binding to ligand or binding partner, inhibiting, triggering, or accelerating biological processes, catalyzing chemical reactions, altering or initiating cell signaling, protein structure (primary, secondary, tertiary, and / or quaternary), and / or chemical properties such as absorbance, fluorescence, buffering, stabilization, solubilization, emulsification and impurity profile.

[0065] As used herein, the term “surfactant” refers to any amphipathic molecule that, when added to water, reduces the surface tension thereof.

[0066] As used herein, the term “substituted” means that the specified group(s) or moiety or moieties bears one or more substituent.

[0067] As used herein, the term “weight average molecular weight" has unit of g / mol or Dalton (Da). The "weight average molecular weight" is preferably determined via Gel Permeation Chromatography (GPC) and / or Light Scattering (e g., ASTM D4001).

[0068] The adherent cells can be animal -derived cells and are preferably mammalian-derived cells. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, marmots, dogs and cats. The adherent cells may be cells derived from the skin, liver, kidneys, muscles, bone, blood vessels, blood, or tissue such as nerve tissue. A single type of cell is typically cultured independently, but a combination of two or more types of cells may also be cultured.

[0069] The molar equivalents of hydroxyl groups in reactant A, an aliphatic hydrophilic polyol or an aliphatic hydrophilic polyether polyol, can be determined by multiplying the moles of reactant A by the number of hydroxyl groups per molecule of reactant A. The molar equivalents of epoxy groups in reactant B, a hydrophobic compound having an epoxy group, can be determined by multiplying the moles of reactant B by the number of epoxy groups per molecule of reactant B.

[0070] The present invention provides a lysis agent for a cell comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group. Preferably, reactant A is an aliphatic hydrophilic polyether polyol; and reactant B is a hydrophobic compound having an epoxy group.

[0071] In another important non-limiting embodiment, the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 : 1, respectively.

[0072] In another important non-limiting embodiment, the lysing agent for a cell comprises the reaction product of: (i) reactant B : a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol.

[0073] In another important non-limiting embodiment, the reactant C has weight average molecular weight of 100 to 10000 Daltons.

[0074] In another important non-limiting embodiment, the aliphatic hydrophilic polyol is ethylene glycol or a polyalkylene glycol.

[0075] In another important non-limiting embodiment, the polyalkylene glycol is selected from the group consisting of diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol 100, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, polyethylene glycol 600, polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3100, polyethylene glycol 3200, polyethylene glycol 3300, polyethylene glycol 3400, polyethylene glycol 3500, polyethylene glycol 3600, polyethylene glycol 3700, polyethylene glycol 3800, polyethylene glycol 3900, polyethylene glycol 4000, propylene glycol, dipropylene glycol, polypropylene glycol 425, glycerol, diglycerol (polyglycerol-2), triglycerol (polyglycerol-3), tetraglycerol (polyglycerol -4), hexaglycerol (polyglycerol-6), decaglycerol (polyglycerol- 10), and EO / PO block copolymers.

[0076] In another important non-limiting embodiment, the hydrophobic compound having an epoxy group is an alkyl glycidyl ether.

[0077] In another important non-limiting embodiment, the alkyl glycidyl ether is selected from the group consisting of 2-ethylhexyl glycidyl ether, n-butyl glycidyl ether, hexyl glycidyl ether, octyl glycidyl ether, nonyl glycidyl ether, decyl glycidyl ether, undecyl glycidyl ether, dodecyl glycidyl ether, tridecyl glycidyl ether, tetradecyl glycidyl ether, pentadecyl glycidyl ether, hexadecyl glycidyl ether, heptadecyl glycidyl ether, octadecyl glycidyl ether, nonadecyl glycidyl ether, cetyl glycidyl ether, 2-hexyldecyl glycidyl ether, 2-octyldodecyl glycidyl ether, 2-heptylundecyl glycidyl ether, 2- (1,3,3-trimethylbutyl) octyl glycidyl ether, 2-decyltetradecyl glycidyl ether, 2-dodecylhexadecyl glycidyl ether, and 2-tetradecyl octadecyl glycidyl ether.

[0078] In another important non-limiting embodiment, the alkyl glycidyl ether is 2-ethylhexyl glycidyl ether.

[0079] In another important non-limiting embodiment, the reaction product comprises a mixture of mono-functionalized aliphatic hydrophilic polyol and multi-functionalized aliphatic hydrophilic polyol.

[0080] In another important non-limiting embodiment, the reaction product comprises a mixture of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether and aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether.

[0081] In another important non-limiting embodiment, the present application provides a reaction product comprising: (i) from 0.05 % to 40.0 % of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether; and (ii) from 60.0 % to 99.95 % of aliphatic hydrophilic polyol multifunctionalized by alkyl glycidyl ether.

[0082] In another important non-limiting embodiment, the present application provides a reaction product comprising: (i) from 0.05 wt.% to 40.0 wt.% of aliphatic hydrophilic polyol monofunctionalized by alkyl glycidyl ether; and (ii) from 60.0 wt.% to 99.95 wt.% of aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether.

[0083] In another important non-limiting embodiment, the aliphatic hydrophilic polyol is polyethylene glycol and the hydrophobic compound having an epoxy group is 2-ethylhexyl glycidyl ether.

[0084] In another important non-limiting embodiment, the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from about 4: 1 to about 1 :2. Preferably, this ratio is about 1: 1.

[0085] In another important non-limiting embodiment, the lysing agent comprises a reaction product of: (i) reactant A: a polyethylene glycol; and (ii) reactant B: 2-ethylhexyl glycidyl ether; wherein the ratio of molar equivalents of hydroxyl groups in the polyethylene glycol to molar equivalents of epoxy groups in 2-ethylhexyl glycidyl ether is about 1 :1 ; wherein the reaction product comprises a mixture of polyethylene glycol mono-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol di-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tri-functionalized by 2- ethylhexyl glycidyl ether, polyethylene glycol tetra-functionalized by 2-ethylhexyl glycidyl ether, and polyethylene glycol penta-functionalized by 2-ethylhexyl glycidyl ether; and wherein the reaction product has the following generic structure:wherein each C11H22O2 unit is independentlywherein each Ci 1H22O2 unit is attached to the polyethylene glycol core or another C11H22O2 unit by a carbon-oxygen single bond; wherein the sum of all n ’s is an integer ranging from 1 to about 90; m is an integer ranging from 1 to 7; a and b are integers, wherein a + (m x b) ranges from 1 to (5 x m); and Q is an (m + 1) valent alkylene group comprising two to twenty carbon atoms, optionally branched, optionally substituted, and optionally containing ether, oxa or aza moieties; and wherein, if Q = -CH2CH2- and m = 1, the reaction product comprises: polyethylene glycol monofunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 1; polyethylene glycol difunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 2; polyethylene glycol trifunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 3; polyethylene glycol tetrafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 4; and polyethylene glycol pentafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 5.

[0086] In another important non-limiting embodiment, the present application provides a reaction product represented by the following structure:wherein n is an integer ranging from 1 to 90 and a and b are integers, the sum of which ranges from 1 to 4.

[0087] In another important non-limiting embodiment, the present application provides a reaction product represented by the following structure:wherein n is an integer ranging from 1 to 90.

[0088] In another important non-limiting embodiment, the reactant C, an aliphatic hydrophilic alkoxylated alcohol, has the structure set out below:wherein R is a linear or branched alkyl group having from 1 to 20 carbons; n > 1 ; and, for each repeat unit, independently, R1= R2= H, R1= H and R2= CH3, or R1= CH3 and R2= H.

[0089] In another important non-limiting embodiment, the aliphatic hydrophilic alkoxylated alcohol is selected from the group consisting of C6-20 alcohol ethoxylates having from 3 to 225 ethoxylate units.

[0090] In another important non-limiting embodiment, the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof selected from the group of bacterial, yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NS0 (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila , and wherein the yeast cells are selected from the group consisting of S. cerevisiae and P. past oris.

[0091] In another important non-limiting embodiment, the cell lysed is in the form of a cell culture or a pellet or a bead.

[0092] In another important non-limiting embodiment, the lysing agent is used as a surfactant.

[0093] In another important non-limiting embodiment, the lysing agent disrupts at least 60 % of the cells while preserving the protein functionality.

[0094] In another important non-limiting embodiment, the lysing agent disrupts at least 70 % of the cells while preserving the protein functionality.

[0095] In another important non-limiting embodiment, the lysing agent disrupts at least 80 % of the cells while preserving the protein functionality.

[0096] In another important non-limiting embodiment, the lysing agent disrupts at least 90 % of the cells while preserving the protein functionality.

[0097] In another important non-limiting embodiment, the lysing agent disrupts at least 98 % of the cells while preserving the protein functionality.

[0098] In another important non-limiting embodiment, lysing agent is readily or inherently biodegradable as determined by OECD301 or OECD302 test.

[0099] In another important non-limiting embodiment, the present application relates to a cell lysis composition comprising: (a) from 0.01 wt.% to 1.0 wt. % of the lysis agent comprising a reactionproduct of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group; (or) (i) reactant B: a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2; (or) wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4:1 to 1 : 1; (b) cells having a desired protein or peptide; and (c) water, buffer, or a combination thereof.

[0100] In another important non-limiting embodiment, the lysing agent is present in amount of 0.01 wt.%, or 0.05 wt.%, or 0.10 wt.%, or 0.15 wt.%, or 0.20 wt.%, or 0.25 wt.%, or 0.30 wt.%, or 0.35 wt.%, or 0.40 wt.%, or 0.45 wt.%, or 0.50 wt.%, or 0.55 wt.%, or 0.60 wt.%, or 0.65 wt.%, or 0.70 wt.%, or 0.75 wt.%, or 0.80 wt.%, or 0.85 wt.%, or 0.90 wt.%, or 0.95 wt.%, or 1.0 wt.%, or 2.0 wt.%, or 3.0 wt.%, or 4 wt.%, or 5 wt.%, or 6 wt.%, or 7 wt.%, or 8 wt.%, or 9 wt.%, or 10 wt.% of the total composition.

[0101] In another important non-limiting embodiment, the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof.

[0102] In another important non-limiting embodiment, the cell lysed is selected from the group consisting of bacterial, yeast, insect, plant, mammalian and fungi cells.

[0103] In another important non-limiting embodiment, the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NS0 (mouse), and HeLa (human).

[0104] In another important non-limiting embodiment, the insect cells are selected from the group consisting of SF9 and S2 (Drosophila).

[0105] In another important non-limiting embodiment, the yeast cells are selected from the group consisting of S. cerevisiae and P. pastoris.

[0106] In another important non-limiting embodiment, the present application relates to a method for lysing a cell comprising the steps of: (a) providing a source of cells; (b) adding a lysis agent comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group; (or) (i) reactant B: a hydrophobic compoundhaving an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000 Daltons; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2; (or) wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4:1 to 1 : 1; and (c) mixing at a temperature of 4 °C to 90 °C.

[0107] In another important non-limiting embodiment, the present application relates to a process for producing a biological product, comprising the steps of: (a) culturing host cells producing the biological product; (b) lysing the cells by a process comprising (bl) providing a source of cells; (b2) adding a lysing agent comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group; (or) (i) reactant B: a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000 Daltons; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1:2; (or) wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 : 1; and (b3) mixing at a temperature of 4 °C to 90 °C; and (c) recovering the biological product.

[0108] In another important non-limiting embodiment, the biological product is selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

[0109] In another important non-limiting embodiment, the present application relates to a process for inactivating an enveloped virus in a biological product comprising contacting the enveloped virus with an amount of at least 0.01 wt.% of lysing agent sufficient to inactivate the virus while recovering the biological product selected from antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

[0110] Further, certain aspects of the present application are illustrated in detail by way of the following examples. The examples are given herein for illustration of the application and are not intended to be limiting thereof.EXAMPLES

[0111] The synthetic scheme reaction of PEG and EHGEwherein each C11H22O2 unit is independentlywherein each C11H22O2 unit is attached to the polyethylene glycol core or another C11H22O2 unit by a carbon-oxygen single bond; wherein the sum of all n ’s is an integer ranging from 1 to about 90; m is an integer ranging from 1 to 7; a and b are integers, wherein a + (m x b) ranges from 1 to (5 x m); and Q is an (m + 1) valent alkylene group comprising two to twenty carbon atoms, optionally branched, optionally substituted, and optionally containing ether, oxa or aza moieties; and wherein, if Q = -CH2CH2- and m = 1, the reaction product comprises: polyethylene glycol monofunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 1; polyethylene glycol difunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 2; polyethylene glycol trifunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 3; polyethylene glycol tetrafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 4; and polyethylene glycol pentafunctionalized by 2-ethylhexyl glycidyl ether, having a + b = 5.

[0112] Experimental Procedure 1 (batch procedure): The reaction of PEG and EHGE was run using the following one-pot procedure: Polyethylene glycol, 2-ethylhexyl glycidyl ether, and potassium hydroxide were combined in a vessel, which was sealed and maintained under a nitrogen atmosphere. The reaction mixture was stirred and heated to 100 °C for 7-19 hours and then cooled to room temperature. The following examples were run using this procedure.

[0113] Example 1: The reaction of PEG 400 (28.50 g), 2-ethylhexyl glycidyl ether (6.64 g), and potassium hydroxide (200 mg) was run for 19 hours at 100 °C following Experimental Procedure 1.

[0114] Example 2: The reaction of PEG 400 (11.00 g), 2-ethylhexyl glycidyl ether (20.49 g), and potassium hydroxide (77 mg) was run for 19 hours at 100 °C following Experimental Procedure 1.

[0115] Example 4: The reaction of PEG 300 (111.51 g), 2-ethylhexyl glycidyl ether (138.49 g), and potassium hydroxide (1.043 g) was run for 7 hours at 100 °C following Experimental Procedure 1.

[0116] Experimental Procedure 2 (slow addition procedure): The reaction of PEG and EHGE was run using the following slow-addition procedure: Polyethylene glycol and potassium hydroxide were combined in a vessel, which was sealed and maintained under nitrogen atmosphere. The reaction mixture was stirred and heated to 100 °C. Once temperature reached 100 °C, 2-ethylhexyl glycidyl ether was slowly added over ~1 hour. The reaction mixture was stirred at 100 °C for 30 minutes and then at 120 °C for 3 to 3.5 hours and then cooled to room temperature. The following examples were run using this procedure.

[0117] Example 3: The reaction of PEG 200 (87.00 g), 2-ethylhexyl glycidyl ether (162.07 g), and potassium hydroxide (1.22 g) were run following Experimental Procedure 2.

[0118] Example 5: The reaction of PEG 400 (520.00 g), 2-ethylhexyl glycidyl ether (484.35 g), and potassium hydroxide (3.65 g) were run following Experimental Procedure 2.

[0119] Example 6: The reaction of PEG 600 (160.00 g), 2-ethylhexyl glycidyl ether (99.35 g), and potassium hydroxide (748 mg) was run following Experimental Procedure 2.

[0120] Example 7: The reaction of PEG 1000 (190.00 g), 2-ethylhexyl glycidyl ether (70.79 g), and potassium hydroxide (533 mg) were run following Experimental Procedure 2.

[0121] Example 8: The reaction of PEG 2000 (210.50 g), 2-ethylhexyl glycidyl ether (39.21 g), and potassium hydroxide (398 mg) was run following Experimental Procedure 2.

[0122] Example 9: The reaction of PEG 4000 (228.50 g), 2-ethylhexyl glycidyl ether (21.28 g), and potassium hydroxide (396 mg) were run following Experimental Procedure 2.

[0123] Reaction products of examples 1 to 9 are neutralized to pH = 5-8 after reaction using either acetic acid or formic acid. The resulting neutralized reaction products are used in cell lysis applications.

[0124] LCMS: LCMS was used to separate mixtures of substituted PEGs, and to determine the relative abundance of PEGs with varying degrees of substitution. Peaks corresponding tounsubstituted PEG, monosubstituted PEG, di substituted PEG, tri substituted PEG and tetrasubstituted PEG were well resolved by HPLC and were thus reliably integrated.

[0125] Procedure: About 0.1 g of each sample was combined with 10 mL of methanol and vortexed until the resulting stock solution was homogenous. For LCMS analysis, 100 pL of stock solution was diluted with 900 pL of methanol, and the resulting solution was injected directly into the system. LCMS analyses were performed on an Agilent 6520 QTOF LC / MS with Agilent 1260 HPLC and MassHunter software. HPLC conditions (Table 1) and MS conditions (Table 2) are tabulated below:Table 1: HPLC ConditionsColumn Agilent Poroshell- 120 SB-C18 2.1x75mm, 2.7pmMobile phase A 10 ruM ammonium acetate in waterMobile phase B 10 mM ammonium acetate in methanol GradientPost run time 15 minFlow rate 0.25 mL / minColumn temperature 40 °CInjection volume2 pLTable 2: MS Conditions

[0126] Table 3 lists the product distributions determined for the reaction products of Examples 1 -7.Table 3: Product Distribution of Substituted PEGs Determined by LCMS, Area %

[0127] Contact angle, static and dynamic surface tension, and foaming measurements of surfactants

[0128] Solution Preparation: Aqueous solutions of surfactants were prepared by combining 1 g of adduct with 999 g of DI water (filtered by Millipore-Q, pH 6.3 to 6.7) at room temperature. The resulting solution was mixed using a magnetic stirrer for 24 hours before further measurement. The solution pH values for select samples were measured to be 7.4.

[0129] Contact angle measurements: Clear, self-adhesive, 0.002-inch-thick polypropylene film (McMaster-Carr # 5577149-01) was used as the substrate to measure the contact angle of 0.1 wt.% aqueous solutions of surfactants.

[0130] Drop Shape Analysis System DSA 305 from Kruss was used to determine contact angle values between a sample drop and polypropylene surface. Sample drops were dispensed automatically using a disposable 1-mL syringe. The contact angle of each drop was determined automatically by the instrument fitting model software (Kruss Advance Drop Shape V 1.5.1) and recorded every second over a 30-second period. Multiple drops were placed at sufficient distances on the pre-cleaned polypropylene surface, and results are reported as average values. At least 6 drops were analyzed for each sample. All measurements were carried out at ambient temperature and relative humidity.

[0131] Static surface tension measurements: Static surface tension values of freshly mixed 0.1 wt.% aqueous solutions of surfactants were measured at room temperature using force tensiometry (Attension, Biolin Scientific) with Wilhelmy Plate method based on the instrument company recommendations (in compliance with ASTM D1331-20). Three surface tension measurements wererecorded for each sample, and average values are reported. The Wilhelmy plate was rinsed and pyrolyzed with flame before every measurement. Calibration of the equipment was checked by measuring the surface tension of DI water at the beginning of measurements.

[0132] Dynamic surface tension measurements: A bubble pressure tensiometer (Kruss, model BP2) was used to measure the dynamic surface tension of freshly mixed 0.1 wt.% aqueous solutions of surfactants between 10 and 100,000 millisecond surface age, utilizing a 0.256 mm-diameter, hydrophobically coated, glass capillary. The capillary was cleaned between samples with detergent solution and rinsed with DI water before drying with Kimwipes. Calibration of the equipment was checked by measuring the surface tension of DI water before experimental measurements.

[0133] Foaming index determination: A SITA Messtechnik foam tester R-2000 was used to evaluate foaming behavior of the surfactants. Approximately 900 mL of freshly mixed 0.1 wt.% solution was fdled in the instrument reservoir, and the test parameters were entered using the instrument software. Each sample was tested 3 times at room temperature. For foam build up, 250 mL of solution was filled in the cylindrical glass vessel and mixed with a bottom propeller for 10 seconds at 800 rpm. After mixing cycle, the foam height formed above the liquid was measured by the conductive probes of the instrument and foam volume was recorded in mL by the instrument software. This step was repeated 30 times and cumulative foam build-up was recorded for 30 consecutive mixing steps. After completing three runs for each sample, the average foam volume for each mixing step was calculated. The foaming index is defined as the total foam volume at the end of 30-mixing steps and was calculated by summing the average values of foam volumes for each mixing step using Excel software (Table 4).Table 4: Contact Angle, Static and Dynamic Surface Tension, and Foaming Measurements*PP = Polypropylene“Control Cl = Lutensol® XP80“Control C2 = Novel® 8-7 EthoxylateEXAMPLES FOR CELL LYSIS

[0134] Reaction product of PEG and EHGE as a membrane lysis agent:

[0135] Example 10: Determine the cytotoxicity of example 4 (300) on an adherent culture of mammalian cells

[0136] Purpose: Demonstrate the ability to lyse mammalian cells using example 4 resulting in low cell viability.

[0137] Cell Type: A549 Cells - A549 cells were isolated from the lung tissue of a White, 58-year- old male with lung cancer. This cell line can be used in cancer, immuno-oncology, and toxicology research.

[0138] Sample Preparation: Reaction products of PEG and EHGE solutions were prepared in DI water such that the final concentrations in the well would be 1%, 0.5%, 0.25%, 0.1% and, 0.05%, as lOuL would be added to lOOuL the resulting concentrations were 1 IX this concentration.

[0139] Analysis Method: a) 15,000 A549 cells (P8, DMEM GlutaMAX, 10% FBS) seeded in 96-well plate. b) Test substance was tested at 1 %, 0.5 %, 0.25 %, 0.1 %, 0.05 % (n=5) concentrations and incubated in 5% CO2 incubator at 37 °C for 18 hours. c) After 18 hours, 10 pL of CCK-8 reagent was added to each well and incubated in a 5% CO2 incubator at 37 °C for 3.5 hours. d) Absorbance was measured at 450 nm.

[0140] Data shown as mean ± SEM, n = 5 and results are depicted in Figure 1, Figure 2 and Figure 3.

[0141] Example 4 exhibited <1% cel viability at each concentration tested (1 %, 0.5 %, 0.25 %, 0.1 %, 0.05 %) with n=5 replicates for each sample concentration. The material investigated was toxic to A549 cells at the lowest concentrations

[0142] Interpretation:

[0143] The detergent properties of example 4 allow it to attack the membrane of A549 cells and lyse them. FIGURE 1 shows the diminished cell viability of A549 cells after exposure to the reaction product of PEG and EHGE at each concentration. FIGURE 2 shows the cells prior to treatment and FIGURE 3 shows post treatment, where the cells have detached from the well and lost all viability.

[0144] Example 11: Determine the ability of Example 7 and 9 (1000 and 3350) to lyse cells while preserving protein functionality

[0145] Purpose: Demonstrate the ability to lyse mammalian cells using example 7 and / or example 9 resulting in low cell viability, while retaining the activity of a target protein that has been transfected into host cells, or demonstration EGFP will be used. Benchmark against industry standard, Triton X-100.

[0146] Cell Type: A549 Cells - A549 cells were isolated from the lung tissue of a White, 58-year- old male with lung cancer. This cell line can be used in cancer, immuno-oncology, and toxicology research.

[0147] Sample Preparation: Solutions were prepared in DI water of each example 7 and example 9 such that the final concentrations in the well would be 0.5%, 0.05% and, 0.01%, as lOuL would be added to lOOuL the resulting concentrations were 1 IX this concentration.

[0148] Analysis Method: a) 20,000 A549 cells (Pl 5, DMEM GlutaMAX, 10% FBS) seeded in 96-well plate and incubated in 5% CO2 incubator at 37 °C for 24 hours. b) Transfection was performed following the Lipofectamine MessengerMAX protocol (0.15 uL MessengerMax, 100 ng EGFP mRNA) and incubated in 5% CO2 incubator at 37 °C for 24 hours. c) After 24 hours, EGFT fluorescence was measured by plate reader (ex.: 495 nm, em.: 519 nm) and images were taken. d) Test substances were tested at 0.5, 0.05, 0.01 %, blk (n=3) and incubated in 5% CO2 incubator at 37 °C. e) After 10 min, 2.5 h and 24 h, images were taken (Keyence) and EGFT fluorescence was measured by plate reader (ex.: 495 nm, em.: 519 nm). f) Only after 24 h cell viability was determined.g) Data shown as mean ± SEM, n = 5 h) Fluorescence microscope: Keyence BZ-X810 i) Plate reader: Perkin Elmer EnSight; Software: Kaleido 3.0 j) Lipofectamine MessengerMAX obtained from ThermoFisher Lot: 2840455 k) Mammalian cell lines used: A549: Passage 15. Obtained at P2 from UC Berkeley Cell Culture Facility. l) Propagated in DMEM GlutaMAX with 10% FBS.

[0149] Results;

[0150] Cells were able to be transfected and express EGFP, prior to lysis. Once exposed to Triton X-100, example 7, and example 9, all cultures showed some level of lysis. The fluorescence intensity was recorded for each case before and after lysis.

[0151] Interpretation:

[0152] The level of lysis observed for Triton X-100 and example 7 demonstrated the same level of cellular disruption with the fluorescence intensity before and after lysis remaining the same indicating there was no interference with the function of the target protein being expressed. Example 9 also demonstrates cellular disruption with the fluorescence intensity before and after lysis remaining the same

[0153] Example 12: Determine the ability of examples 7 and 9 (1000 and 3350) to lyse cells

[0154] Purpose: Demonstrate the ability to lyse insect cells using examples 7 and / or example 9 resulting in low cell viability. Benchmark against industry standard, Triton X-100.

[0155] Cell Type: SF-9 Cells - Sf9 is a cell line exhibiting epithelial morphology that was derived from pupa ovarian tissue of a fall armyworm. This cell line can be used to replicate baculovirus expression vectors.

[0156] Sample Preparation: Solutions were prepared in DI water of each example 7 and example 9 such that the final concentrations in the sample holder would be 0.5%, 0.05% and, 0.01% using a 2% stock and diluting to the final volume with buffer.

[0157] Analysis Method:

[0158] 0.01-0.5 w / w% lysis agent was added to 7.48 x I CP SF-9 cells (EX-CELL® 420 Serum Medium) seeded into a centrifuge tube. After 10 minutes exposure; cells were stained with trypan blue (live / dead stain) and counted using a cell counter to quantify living cells (Table 5).Table 5: Cell viability after exposure

[0159] Results:

[0160] Insect cells were successfully cultured and demonstrated viability at 7.48 x 105. Exposed insect cells were successfully stained and could be imaged with live / dead stain and a count of viable cells taken. The exposed cells from Triton X-100 and Example 7 had similar reduction in viable cell counts. The exposed cells from Example 9 had lowered reduction in viable cell counts, leaving a higher number of viable cells.

[0161] Interpretation:

[0162] Examples 7 and 9 can be used as a detergent strong enough to lyse insect cells. Example 7 lyses cells to a similar level as TritonX-100, is inherently biodegradable, and does not form toxic byproducts and can be a suitable replacement for Triton X -100.

[0163] Example 13: Red blood cell lysis

[0164] Bovine red blood cells (Innovative Research) were diluted into 0.1M phosphate buffer pH 7.4 50X in falcon tubes and mixed gently to evenly suspend them. Samples were added to sterile 96- well microplates (10 pL each) in triplicate. Diluted red blood cells were then added to each well(190 JJ.L). Microplates were incubated at 37 °C in a shaker incubator for 1 hour. Microplates were next centrifuged at 500 x g and 15 °C for 5 minutes, then 100 pL of the supernatant was transferred to clear 96-well microplates. Absorbance of hemoglobin released from lysed cells was measured at 540 nm using an Agilent Biotek Synergy Hl Microplate reader. Average absorbance of the cell supernatant without additives was subtracted from the average absorbance of the samples. Absorbance values are shown in Figure 24.

[0165] Bovine hemoglobin was dissolved in 0. IM phosphate buffer pH 7.4 at 5 mg / mL. Hemoglobin (190 uL) and surfactant (10 uL) stock solutions were added to a 96-well microplate and incubated at 37 °C in a shaker incubator for 1 hour. A photograph was taken to visualize hemoglobin stability in the presence of surfactants. Photographs are shown in Figure 25.

[0166] Interpretation:

[0167] Red blood cells (RBC) were used to evaluate lysing agents effect on mammalian cells. Release of hemoglobin protein after exposure to lysis agents was used to evaluate performance. All PEG-EHGE lysis agents were effective at RBC lysis at 1% concentration to a similar level as Triton X-100. PEG1000-1 EHGE, PEG1000-2 EHGE, PEG1000-3 EHGE, and Triton X-100 were similarly effective at RBC lysis at 0.1%. PEG1000-2 EHGE, PEG1000-3 EHGE were more effective at RBC lysis than Triton X-100 at 0.01%. Cell lysis agents were added to hemoglobin protein to evaluate the effect on protein stability. Hemoglobin did not aggregate in the presence of surfactants.

[0168] RVLP reduction assay

[0169] A solution of 1% bovine serum albumin (BSA) was prepared in phosphate buffered saline (PBS) pH 7.4. Retrovirus-like particles (RVLP, Cygnus) were added into this solution to a final concentration of 1% to prepare an RVLP stock solution. Surfactant was added to aliquots of the RVLP stock solution for a final concentration of 0.5, 0.1, 0.01 and 0% surfactant. The samples were mixed then incubated at room temperature (22 °C) for 60 minutes. Surfactant was removed using HiPPR detergent removal columns (ThermoFisher) following the manufacturer's instructions. Samples were frozen at -80 °C until analysis. RVLP inactivation was done using the MockV RVLP Inactivation Kit, Extraction Kit, and qPCR kits (Cygnus) following the manufacturer's instructions. Log reduction value (LRV) was calculated relative to the 0% surfactant sample. The results are shown in Figure 26.

[0170] Steps to ensure clearance of adventitious and endogenous viruses are required during production of biopharmaceuticals. Addition of detergents / surf actants is a common method to inactivate and reduce enveloped viruses. Triton X-100 has been used as a detergent for viral inactivation during bioprocessing (eg. ASTM E3042-16). Retrovirus-like particles (RVLP) are enveloped particles endogenous to Chinese hamster ovary (CHO) cell culture, which is commonly used for production of biopharmaceuticals. ICH Q5A(R2) guidelines for viral safety evaluation state that CHO-derived endogenous virus particles can be used to demonstrate viral clearance. The viral inactivation properties of PEG-EHGE surfactants were tested using RVLP. At 0.5%, PEG1000-2 EHGE and PEG1000-3 EHGE result in RVLP produce similar log reduction in RVLP compared to the same concentration of Triton X-100. At 0.1 and 0.01%, PEG1000-2 EHGE and PEG1000-3 EHGE reduce RVLP to a greater extent than Triton X-100 at the same concentrations. Both PEG1000-2 EHGE and PEG1000-3 EHGE have comparable or improved viral clearance characteristics compared to Triton X-100.

[0171] N-Dhenyl-l-napthylamine CMC assay

[0172] A stock solution of N-phenyl-l-napthylamine (NPN) was prepared at 5 mg / mL in ethanol. The fluorescent dye solution (0.15 M sodium chloride, 0.05 M TRIS, 5 pMNPN, and 15 ppm Brij 35, pH 8.0) was then prepared and fdtered through a 0.22 um bottle top fdter. Serial dilutions of surfactant solutions were prepared in a black-walled 96-well microplate. Fluorescence was measured (excitation 3 0 nm, emission 420 nm). Fluorescence intensity was background corrected from wells that contained only dye solution. Corrected fluorescence intensity was plotted versus surfactant concentration, and a polynomial fit was applied. The inflection point (critical micelle concentration (CMC) was determined from the y-intercept of the second derivative of the polynomial fit. CMC values for various test articles are tabulated below.Table 6: Test Articles and their Critical Micelle ConcentrationTest article Critical micelle n (%)

[0173] Example 14: BIODEGRADATION

[0174] Biodegradation of surfactants as per OECD301F standard test with Benzoate at experimental control details are provided in figure 28.Table 7: Biodegradation Studies

[0175] Embodiment 1: A lysing agent for a cell comprising a reaction product of: (i) reactant A: an aliphatic hydrophilic polyol; and (ii) reactant B: a hydrophobic compound having an epoxy group; (or) (i) reactant B: a hydrophobic compound having an epoxy group; and (ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000 Daltons; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2; (or) wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 : 1.

[0176] Embodiment 2: The lysing agent according to embodiment 1, the lysing agent is readily or inherently biodegradable as determined by OECD301 or OECD302 test.

[0177] Embodiment 3: The lysing agent according to embodiment 1, wherein the aliphatic hydrophilic polyol is ethylene glycol or a polyalkylene glycol selected from the group consisting of di ethylene glycol, tri ethylene glycol, tetraethylene glycol, polyethylene glycol 100, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, polyethylene glycol 600, polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400,polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3100, polyethylene glycol 3200, polyethylene glycol 3300, polyethylene glycol 3400, polyethylene glycol 3500, polyethylene glycol 3600, polyethylene glycol 3700, polyethylene glycol 3800, polyethylene glycol 3900, polyethylene glycol 4000, propylene glycol, dipropylene glycol, polypropylene glycol 425, glycerol, diglycerol (polyglycerol-2), triglycerol (polyglycerol-3), tetraglycerol (polyglycerol-4), hexaglycerol (polyglycerol-6), decaglycerol (polyglycerol- 10), and EO / PO block copolymers, and the hydrophobic compound having an epoxy group is an alkyl glycidyl ether selected from the group consisting of 2-ethylhexyl glycidyl ether, n-butyl glycidyl ether, octyl glycidyl ether, nonyl glycidyl ether, decyl glycidyl ether, undecyl glycidyl ether, dodecyl glycidyl ether, tridecyl glycidyl ether, tetradecyl glycidyl ether, pentadecyl glycidyl ether, hexadecyl glycidyl ether, heptadecyl glycidyl ether, octadecyl glycidyl ether, nonadecyl glycidyl ether, cetyl glycidyl ether, 2-hexyldecyl glycidyl ether, 2-octyldodecyl glycidyl ether, 2-heptylundecyl glycidyl ether, 2-(l,3,3- trimethylbutyl) octyl glycidyl ether, 2-decyltetradecyl glycidyl ether, 2-dodecylhexadecyl glycidyl ether, and 2-tetradecyloctadecyl glycidyl ether.

[0178] Embodiment 4: The lysing agent according to embodiment 1, wherein the reaction product comprises (i) a mixture of mono-functionalized aliphatic hydrophilic polyol and multifunctionalized aliphatic hydrophilic polyol, (or) (ii) a mixture of aliphatic hydrophilic polyol monofunctionalized by alkyl glycidyl ether and aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether.

[0179] Embodiment 5: The lysing agent according to embodiment 4, wherein the reaction product comprises: (i) from 0.05% to 40.0% of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether; and (ii) from 60.0% to 99.95% of aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether.

[0180] Embodiment 5A: The lysing agent according to embodiment 4, wherein the reaction product comprises: (i) from 0.05 wt.% to 40.0 wt.% of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether; and (ii) from 60.0 wt.% to 99.95 wt.% of aliphatic hydrophilic polyol multifunctionalized by alkyl glycidyl ether.

[0181] Embodiment 6. The lysing agent according to embodiment 1 , wherein the aliphatic hydrophilic polyol is polyethylene glycol and the hydrophobic compound having an epoxy group is 2-ethylhexyl glycidyl ether, and wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2.

[0182] Embodiment 7. A lysing agent for a cell comprising a reaction product of: (i) reactant A: a polyethylene glycol; and (ii) reactant B: 2-ethylhexyl glycidyl ether; wherein the ratio of molar equivalents of hydroxyl groups in the polyethylene glycol to molar equivalents of epoxy groups in 2-ethylhexyl glycidyl ether is about 1 :1; wherein the reaction product comprises a mixture of polyethylene glycols mono-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol difunctionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tri -functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tetra-functionalized by 2-ethylhexyl glycidyl ether, and polyethylene glycol penta-functionalized by 2-ethylhexyl glycidyl ether; wherein the reaction product has the following generic structure:? wherein each C11H22O2 unit is independentlywherein each C11H22O2 unit is attached to the polyethylene glycol core or another C11H22O2 unit by a carbon-oxygen single bond; wherein the sum of all n ’s is an integer ranging from 1 to 90; m is an integer ranging from 1 to 7; a and b are integers, wherein a + (m x b) ranges from 1 to (5 x m); and Q is an (m + l)-valent alkylene group comprising two to twenty carbon atoms, optionally branched, optionally substituted, and optionally containing ether, oxa or aza moieties; and wherein, if Q = -CH2CH2- and m = 1, the reaction product comprises: polyethylene glycol mono-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 1; polyethylene glycol di-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 2; polyethylene glycol tri-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 3; polyethylene glycol tetra-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 4; andpolyethylene glycol penta-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 5.

[0183] Embodiment 8. The lysing agent according to embodiment 7, wherein the reaction product is represented by the following structure:wherein n is an integer ranging from 1 to 90 and a and b are integers, the sum of which ranges from 1 to 4.

[0184] Embodiment 9. The lysing agent according to embodiment 8, wherein the reaction product is represented by the following structure:wherein n is an integer ranging from 1 to 90; wherein the reactant has a weight average molecular weight in the range of 1000 to 5000 Daltons.

[0185] Embodiment 10. The lysing agent according to embodiment 1, wherein the reactant C: an aliphatic hydrophilic alkoxylated alcohol, has the structure set out below:wherein R is a linear or branched alkyl group having from 1 to 20 carbons; n > 1; and, for each repeat unit, independently, R1= R2= H, R1= H and R2= CH3, or R1= CH3 and R2= H.

[0186] Embodiment 11. The lysing agent according to embodiment 10, wherein the aliphatic hydrophilic alkoxylated alcohol is selected from the group consisting of C6-20 alcohol ethoxylates having from 3 to about 225 ethoxylate units.

[0187] Embodiment 12. The lysing agent according to embodiment 1, wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof selected from the group of bacterial,yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila , and wherein the yeast cells are selected from the group consisting of S. cerevisiae and P. pastoris.

[0188] Embodiment 13. The lysing agent according to embodiment 1, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

[0189] Embodiment 14. The lysing agent according to embodiment 1, wherein the lysing agent disrupts at least 60% of the cellular membranes while preserving the protein functionality.

[0190] Embodiment 15. A cell lysis composition comprising: (a) from 0.01 wt.% to 0.5 wt.% of the lysing agent of embodiment 1; (b) cells having a desired protein or peptide; and (c) water, buffer, or a combination thereof; wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof; selected from the group of bacterial cells, fungi cells, insect cells, mammalian cells, plant cells, and yeast cells; wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila}, wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); and wherein the yeast cells are selected from the group consisting of S. cerevisiae and P. pastoris.

[0191] Embodiment 16. The cell lysis composition according to Embodiment 15, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

[0192] Embodiment 17. A method for lysing a cell comprising the steps of: (a) providing a source of cells; (b) adding a lysing agent according to embodiment 1; and (c) mixing at a temperature of 4 °C to 90 °C.

[0193] Embodiment 18. The method for lysing a cell according to embodiment 17, wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof selected from the group of bacterial, yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila and wherein the yeast cells are selected from the group consisting of S. cerevisiae and P. pastoris.

[0194] Embodiment 19. The method for lysing a cell according to embodiment 18, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

[0195] Embodiment 20. A process for producing a biological product, comprising the steps of: (a) culturing host cells producing biological products; (b) lysing the cells by a process according to embodiment 17; and (c) recovering the biological product selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

[0196] Embodiment 21. A process for inactivating an enveloped virus in a biological product comprising contacting the enveloped virus with an amount of at least 0.01 % to 0.5 wt.% of lysing agent according to embodiment 1 sufficient to inactivate the virus while recovering the biological product selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

[0197] While the compositions and methods of the disclosed and / or claimed inventive concept(s) have been described in terms of particular aspects, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosed and / or claimed inventive concept(s). All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosed and / or claimed inventive concept(s).

Claims

Patent Claims:

1. A lysing agent for a cell comprising a reaction product of(i) reactant A: an aliphatic hydrophilic polyol; and(ii) reactant B: a hydrophobic compound having an epoxy group; or(i) reactant B: a hydrophobic compound having an epoxy group; and(ii) reactant C: an aliphatic hydrophilic alkoxylated alcohol, having a weight average molecular weight of 100 to 10000 Daltons; wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2; or wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 : 1.

2. The lysing agent according to claim 1, wherein the lysing agent is readily or inherently biodegradable as determined by OECD301 or OECD302 test.

3. The lysing agent according to claim 1, wherein the aliphatic hydrophilic polyol is ethylene glycol or a polyalkylene glycol, which is preferably selected from the group consisting of diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol 100, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 500, polyethylene glycol 600, polyethylene glycol 700, polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100, polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800, polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3100, polyethylene glycol 3200, polyethylene glycol 3300, polyethylene glycol 3400, polyethylene glycol 3500, polyethylene glycol 3600, polyethylene glycol3700, polyethylene glycol 3800, polyethylene glycol 3900, polyethylene glycol 4000, propylene glycol, dipropylene glycol, polypropylene glycol 425, glycerol, diglycerol (polyglycerol-2), triglycerol (polyglycerol-3), tetraglycerol (polyglycerol-4), hexaglycerol (polyglycerol-6), decaglycerol (polyglycerol -10), and EO / PO block copolymers, and the hydrophobic compound having an epoxy group is an alkyl glycidyl ether, which is preferably selected from the group consisting of 2-ethylhexyl glycidyl ether, n-butyl glycidyl ether, octyl glycidyl ether, nonyl glycidyl ether, decyl glycidyl ether, undecyl glycidyl ether, dodecyl glycidyl ether, tridecyl glycidyl ether, tetradecyl glycidyl ether, pentadecyl glycidyl ether, hexadecyl glycidyl ether, heptadecyl glycidyl ether, octadecyl glycidyl ether, nonadecyl glycidyl ether, cetyl glycidyl ether, 2-hexyldecyl glycidyl ether, 2-octyldodecyl glycidyl ether, 2-heptylundecyl glycidyl ether, 2-( 1,3, 3 -trimethylbutyl) octyl glycidyl ether, 2-decyltetradecyl glycidyl ether, 2- dodecylhexadecyl glycidyl ether, and 2-tetradecyloctadecyl glycidyl ether.

4. The lysing agent according to claim 1 or claim 3, wherein the reaction product comprises(i) a mixture of mono-functionalized aliphatic hydrophilic polyol and multifunctionalized aliphatic hydrophilic polyol, or(ii) a mixture of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether and aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether.

5. The lysing agent according to claim 4, wherein the reaction product comprises:(i) from 0.05 % to 40.0 % of aliphatic hydrophilic polyol mono-functionalized by alkyl glycidyl ether, preferably based on the total weight of the lysing agent and / or the reaction product; and(ii) from 60.0 % to 99.95 % of aliphatic hydrophilic polyol multi-functionalized by alkyl glycidyl ether, preferably based on the total weight of the lysing agent and / or the reaction product.

6. The lysing agent according to any of the preceding claims, wherein the aliphatic hydrophilic polyol is polyethylene glycol and the hydrophobic compound having an epoxy group is2-ethylhexyl glycidyl ether, and wherein the ratio of molar equivalents of hydroxyl groups in reactant A to molar equivalents of epoxy groups in reactant B is from 4: 1 to 1 :2.

7. A lysing agent for a cell comprising a reaction product of:(i) reactant A: a polyethylene glycol; and(ii) reactant B: 2-ethylhexyl glycidyl ether; wherein the ratio of molar equivalents of hydroxyl groups in the polyethylene glycol to molar equivalents of epoxy groups in 2-ethylhexyl glycidyl ether is about 1 : 1, such as from 1.5: 1 to 1 : 1.5; wherein the reaction product comprises a mixture of polyethylene glycol mono-functionalized by 2- ethylhexyl glycidyl ether, polyethylene glycol di-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tri-functionalized by 2-ethylhexyl glycidyl ether, polyethylene glycol tetrafunctionalized by 2-ethylhexyl glycidyl ether, and polyethylene glycol penta-functionalized by 2- ethylhexyl glycidyl ether; wherein the reaction product has the following generic structure:•> wherein each C11H22O2 unit is independentlywherein each C11H22O2 unit is attached to the polyethylene glycol core or another C11H22O2 unit by a carbon-oxygen single bond; wherein the sum of all n ’s is an integer ranging from 1 to 90; m is an integer ranging from 1 to 7; a and b are integers, wherein a + (m x b) ranges from 1 to (5 x m) and Q is an (m + l)-valent alkylene group comprising two to twenty carbon atoms, optionally branched, optionally substituted, and optionally containing ether, oxa or aza moieties; and wherein, if Q = -CH2CH2- and m = 1, the reaction product comprises: polyethylene glycol mono-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 1; polyethylene glycol di-functionalized by 2-ethylhexyl glycidyl ether, having cz + Z> = 2;polyethylene glycol tri-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 3; polyethylene glycol tetra-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 4; and polyethylene glycol penta-functionalized by 2-ethylhexyl glycidyl ether, having a + b = 5.

8. The lysing agent according to claim 7, wherein the reaction product is represented by the following structure:wherein n is an integer ranging from 1 to 90 and a and b are integers, the sum of which ranges from 1 to 4.

9. The lysing agent according to claim 7 or claim 8, wherein the reaction product is represented by the following structure:wherein n is an integer ranging from 1 to 90; wherein the reactant has a weight average molecular weight in the range of 1000 to 5000 Daltons.

10. The lysing agent according to any of the preceding claims, wherein the reactant C: an aliphatic hydrophilic alkoxylated alcohol, has the structure set out below:wherein R is a linear or branched alkyl group having from 1 to 20 carbons; n > 1; and, for each repeat unit, independently, R1= R2= H, R1= H and R2= CH3, or R1= CH3 and R2= H.11 . The lysing agent according to claim 10, wherein the aliphatic hydrophilic alkoxylated alcohol is selected from the group consisting of Ce-20 alcohol ethoxylates having from 3 to about 225 ethoxylate units.

12. The lysing agent according to any of the preceding claims, wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof, preferably selected from the group consisting of bacterial, yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila)', and wherein the yeast cells are selected from the group consisting of . cerevisiae and P. pastoris.

13. The lysing agent according to any of the preceding claims, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

14. The lysing agent according to any of the preceding claims, wherein the lysing agent disrupts at least 60% of the cellular membranes while preserving the protein functionality.

15. A cell lysis composition comprising:(a) from 0.01 wt.% to 0.5 wt.% of the lysing agent of any of the preceding claims;(b) cells having a desired protein or peptide; and(c) water, buffer, or a combination thereof; wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof; selected from the group of bacterial cells, fungi cells, insect cells, mammalian cells, plant cells, and yeast cells; wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila), wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); and wherein the yeast cells are selected from the group consisting of . cerevisiae and P. pastoris.

16. The cell lysis composition according to claim 15, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

17. A method for lysing a cell comprising the steps of:(a) providing a source of cells;(b) adding a lysing agent according to any of the preceding claims; and(c) mixing at a temperature of 4 °C to 90 °C.

18. The method for lysing a cell according to claim 17, wherein the cell lysed is a prokaryotic cell, a eukaryotic cell or a combination thereof, preferably selected from the group consisting of bacterial, yeast, insect, plant, mammalian and fungi cells; wherein the mammalian cells are selected from the group consisting of CHO (hamster), HEK293 (human), Sp2 / 0 (mouse), NSO (mouse), and HeLa (human); wherein the insect cells are selected from the group consisting of SF9 and S2 (Drosophila , and wherein the yeast cells are selected from the group consisting of S. cerevisiae and P. pastor is.

19. The method for lysing a cell according to claim 17 or claim 18, wherein the cell lysed is in the form of a cell culture or a pellet or a bead.

20. A process for producing a biological product, comprising the steps of:(a) culturing host cells producing biological products;(b) lysing the cells by a process according to any of the claims 17 to 19; and(c) recovering the biological product selected from the group consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

21. A process for inactivating an enveloped virus in a biological product comprising contacting the enveloped virus with an amount of at least 0.01 wt. % to 0.50 wt.% of lysing agent according to any of the preceding claims, preferably based on the total weight of the biological product, sufficient to inactivate the virus while recovering the biological product selected from thegroup consisting of antibodies, proteins, nucleic acids (genes, vectors), polypeptides, hormones, polynucleotides and viruses.

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