WO2019076955A1

WO2019076955A1 - Immunoassay for bio-active polypeptide stability

Info

Publication number: WO2019076955A1
Application number: PCT/EP2018/078347
Authority: WO
Inventors: Frank Winther Rasmussen
Original assignee: Novozymes A/S
Priority date: 2017-10-18
Filing date: 2018-10-17
Publication date: 2019-04-25

Abstract

The instant invention provides a method for determining the stability of at least one biologically active polypeptide of interest, said method comprising the steps of: • a) providing the at least one biologically active polypeptide; • b) exposing the at least one biologically active polypeptide to one or more conditions of interest for a period of time, whereby the stability of the at least one biologically active polypeptide of interest is challenged; and • c) determining the immunoreactivity of the at least one biologically active polypeptide of interest, wherein the stability of the at least one biologically active polypeptide of interest is determined by its immunoreactivity. Preferably, steps (a) through (c) are repeated at least once and immunoreactivity determinations are converted into Intact Polypeptide Equivalents, IPE. The application shows that the residual immunoreactivity of enzymes correlates very well with the specific functionality as determined by enzyme activity assays. Thus, the otherwise cumbersome process of identifying stabilized enzyme variants by screening them for activity was greatly simplified.

Description

IMMUNOASSAY FOR BIO-ACTIVE POLYPEPTIDE STABILITY

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides methods for determining the stability over time of at least one biologically active polypeptide using an immunoassay.

The art of measuring protein stability, typically, relies on protein functionality, e.g. enzymatic activity for enzymes or ligand binding for receptors, as a measure for protein stability.

Usually, the residual functionality of a protein is measured after being exposed to stress over time and the half-life of the protein can then be determined or calculated. Testing protein variants in parallel to their parent protein in this manner enables screening and selecting, e.g., more or less stable protein variants.

This invention discloses a more general method for determining the stability of proteins; we show that the residual Immunoreactivity of enzymes correlates very well with the specific functionality as determined by enzyme activity assays. Thus, the otherwise cumbersome process of idenfying stabilized enzyme variants by screening the variants for activity was greatly simplified.

The generic immunoreactivity assay of the instant invention also lends itself to higher levels of automation and efficiency; in particular, two assay formats are contemplated:

- a microtiter plate format, wherein the immunoreactivity is measured before and after one or more stress challenge; and

- a microfluidic format, where each microdroplet contains a single variant of a parent protein (or the encoding DNA along with in vitro translation components), and the immunoreactivity of the microdroplets is measured before and after one or more stress challenge to enable sorting of the droplets to provide a pool of droplets enriched for more (or less) stable protein variants.

An antibody capable of binding a biologically active parent polypeptide is provided, which does not bind to a parent polypeptide that has lost its structural integrity, e.g., if it has denatured or otherwise lost its proper folding and activity.

We show herein that the loss of structural integrity of a polypeptide over time correlates very well with the loss of its immunoreactivity. In other words, the structural stability in terms of the amount of antibody that is capable of binding to a polypeptide in an immunoassay is a convenient measure of the biological activity of said polypeptide at any given time.

Multiple immunoassay measurements can be run in parallel for several biologically active polypeptides to provide a convenient way to rank their structural stability over time and under certain conditions and, thus, to select a more (or less) stable polypeptide, as desired. BACKGROUND OF THE INVENTION

The screening of polynucleotide libraries for variants that encode preferred protein activities is a central enterprise in biotechnology (See, e.g. , Jackel and Hilvert, Curr Opin Biotechnol 21 , 753-759 (2010)). Unfortunately, as library diversity increases, there is an exponential decrease in the number of active variants in the library (See, e.g. , Guo et al., PNAS (2004); Bloom et al., PNAS (2005)). This inverse relationship results in inefficient and costly screening of polynucleotide libraries. Since only a small fraction of the library encodes for functional proteins, it is often necessary to screen thousands, millions, or even billions of variants in order to identify desired variants. Screening is often slow and costly because it typically requires custom host cell transformation, protein expression and frequently purification and quantification, specific reaction with substrate or ligand, signal detection and quantitation.

Many methods and extensive robotic automation have been developed to facilitate library screening efforts (See, e.g., Maerkl, Curr Opin Biotechnol 22, 59-65 (201 1 ); Goddard and Reymond, Curr Opin Biotechnol 15, 314-322 (2004); Wahler and Reymond, Curr Opin Biotechnol 12, 535-544 (2001 )). Nonetheless, screening throughput is insufficient and remains costly because promising variants must be identified from a huge pool of possibilities. For example, mutating two random amino acids in a 100-amino acid protein results in a library containing 1.98 x 10⁶ unique members (See, e.g. , Dietrich et al, Ann Rev Biochem 79, 563-590 (2010)). A central limitation of all screening approaches is that they must be customized both to the activity or interaction of the target protein with specific reactants and to the means of detecting and isolating positive variants.

Biological protein activity, temperature stability or storage stability are all factors that are of interest when screening polypeptide variant libraries.

Immunoassays can be performed in multiple different formats; see, for example:

Immunoassay Methods; by: Karen L. Cox, Viswanath Devanarayan, Aidas Kriauciunas, Joseph Manetta, Chahrzad Montrose, and Sitta Sittampalam (May 1 , 2012, Assay Guidance Manual).

A myriad of immunoassay forms and formats have been developed since the first description of the Radioimmunoassay (RIA) back in the late 1950s. The most famous format today is probably the Enzyme-Linked Immunosorbent Assay or ELISA in short.

The general concept of isolating one or more genetic elements encoding a gene product having a desired activity, comprising of the 3 steps of: (a) compartmentalising genetic elements into microcapsules or microdroplets; (b) expressing the genetic elements to produce their respective gene products within the microcapsules/droplets; and (c) sorting the microcapsules or microdroplets to isolate the genetic elements which produce the gene product having the desired activity was described already in 1999 (WO 99/02671 ).

It has also been described to use in vitro expression systems in microcapsules along with an emulsion-stabilizing chemically inert silicone-based surfactant (WO 03/044187). Manipulation of microdroplets, including merger or coalescence of several droplets has been achieved, for example, through the application of an electric field (WO 2007/089541 ).

A number of comprehensive reviews are available and many of the microfluidic components are commercially available. The field of microfluidics is in rapid development and any potential improvements are highly desired.

SUMMARY OF THE INVENTION

Similar to an enzymatic assay, the immunoassay of the instant invention makes it possible to measure the concentration of a biologically active polypeptide, such as, an enzyme, in a sample even if the sample also contains substantial amounts of misfolded, denaturated, degraded or otherwise biologically inactive versions of the same polypeptide. Consequently, the immunoassay can be used for measuring the stability of a biologically active polypeptide over time, without the need of any separation steps. We show herein that the loss of biological activity or functionality of a polypeptide, eg., the loss of the enzymatic activity of an enzyme, is surprisingly accompanied by a comparable loss of immunoreactivity. In this manner, an enzyme's loss of reactivity in an immunoassay of the instant invention over time can be employed as an indirect measure of enzyme stability.

A requirement for carrying out immunoassays is, of course, an antibody that is specific for the biologically active polypeptide to be assayed. A polyclonal antibody may be employed, although one or more monoclonal antibody is also a possibility. Typically, a rabbit polyclonal antibody raised by immunization is used.

In a first aspect, the invention provides a method for determining the stability of at least one biologically active polypeptide of interest, said method comprising the steps of:

a) providing the at least one biologically active polypeptide;

b) exposing the at least one biologically active polypeptide to one or more conditions of interest for a period of time, whereby the stability of the at least one biologically active polypeptide of interest is challenged; and

c) determining the immunoreactivity of the at least one biologically active polypeptide of interest, wherein the stability of the at least one biologically active polypeptide of interest is determined by its immunoreactivity.

In a second aspect the invention the invention relates to the use of a method as defined in the first aspect in a microtiter plate or in a microfluidic format.

DEFINITIONS

Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences. Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276- 277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Variant: The term "variant" means a polypeptide having biological activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions compared to its parent polypeptide. A substitution means replacement of the amino acid occupying a position with a different amino acid in the variant; a deletion means removal of the amino acid occupying a position in the parent polypeptide; and an insertion means adding an amino acid in the variant adjacent to and immediately following the amino acid occupying a position in the parent polypeptide.

Immunoreactivity: The signal of a sample in an immunoassay - typically an ELISA assay.

Target protein: The protein of interest; The protein being studied. Other proteins are not considered. Degraded protein: Protein that has lost its function (enzymatic activity; binding capability etc) - typically due to some structural changes.

PPRAS (Percent protein remaining after stress): Concentration of target protein in sample after stress / Cone, before stress ^*100%. - A measure of how much protein survived the stressful conditions.

Intact protein equivalent. (IPE) : When a sample of a target protein is being stressed, a certain percentage of the target protein degrades. Typically, degradation result in structural changes. Thus, the sample becomes a mixture of intact target protein and structurally altered target protein.

In theory, structurally altered target proteins might contribute to the immunoreactivity of the entire sample. Therefore, the immunoreactivities (Elisa signal) of the samples are expressed as "intact protein equivalents", which means: The dose of "intact protein" which has an equivalent immunoreactivity. However, in practice, a standard curve of different dosages of intact target protein analyzed in the immunoassay can easily be constructed and used for converting sample immunoreactivity signals into IPEs, which can then be used to calculate the PPRAS of the samples. The essence of this invention is the surprising discovery, that degraded target proteins (structurally damaged) in a sample do not contribute significantly to the immunoreactivity of the sample. Therefore, stability can actually be measured in-vitro using an immunoassay rather than a functional (f.ex. enzymatic) assay. In addition, introducing stabilizing substitutions doesn't change anything; stability can still be monitored using an immunoassay.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a method for determining the stability of at least one biologically active polypeptide of interest, said method comprising the steps of:

a) providing the at least one biologically active polypeptide;

A preferred embodiment relates to a method of the first aspect, wherein the at least one biologically active polypeptide is one or more enzyme, preferably selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha- galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta- glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectate lyase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase.

In another preferred embodiment, the at least one biologically active polypeptide consists of a single domain; preferably the at least one biologically active polypeptide consists of at most 1 ,000 amino acids; preferably at most 950 amino acids; more preferably at most 900, 850, 800, 750, 700, 650, 600, 550, or most preferably at most 500 amino acids.

Preferably, the at least one biologically active polypeptide comprises a parent polypeptide and one or more variant thereof, wherein the one or more variant comprises at least one amino acid alteration, such as, a deletion, insertion or substitution of at least one amino acid in one or more positions, and wherein the one or more variant comprises an amino acid sequence at least 70% identical to that of the parent; preferably at least 75% identical, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of the parent; more preferably the method comprises an additional step of identifying and selecting at least one variant having a higher or lower stability over said period of time than its parent.

In a preferred embodiment, the stability comprises one or more of storage stability, temperature stability, structural stability, heat stability, saline stability, and pH stability.

A preferred embodiment relates to a method of the first aspect, wherein the one or more conditions of interest comprise temperature, pH, surfactant concentration, salt concentration, Ca++ concentration, and/or EDTA concentration.

It is preferred that the at least one biologically active polypeptide of interest is comprised in one or more composition; preferably said composition is a laundry detergent composition, an automatic dishwasher detergent composition, a food processing aid composition, a food additive composition, a beverage processing aid composition, a beverage additive composition, a feed processing aid composition, a feed additive composition, a seed-coating composition, an agricultural inoculant composition or an industrial enzyme composition.

Preferably, the method of the first aspect is carried out in a microtiterplate format; or in a microfluidic format wherein the sample of step (a) is comprised in an aqueous microdroplet in an immiscible carrier fluid.

In another preferred embodiment of the first aspect, the immunoreactivity of the at least one biologically active polypeptide of interest is determined in step (c) by comparison with an immunoreactivity standard curve of a reference biologically active polypeptide of interest; preferably the at least one biologically active polypeptide is a variant of a parent polypeptide which is the reference.

Preferably, the immunoreactivity is determined using an antibody raised against the purified biologically active polypeptide of the interest; preferably the antibody is polyclonal; even more preferably the polyclonal antibody is raised in rabbit. Yet another preferrred embodiment relates to the method of the first aspect, wherein steps (a) through (c) are repeated at least once, wherein the immunoreactivity determinations are compared and the polypeptides are ranked, with the polypeptide having the smallest percentage difference between the immunoreactivity measurements as the most stable; preferably, the immunoreactivity determinations are converted into Intact Polypeptide Equivalents, IPE, using an immunoreactivity standard curve constructed using the parent polypeptide or a variant thereof, wherein %-residual IPE is calculated for each biologically active polypeptide of interest, and wherein the biologically active polypeptide of interest which is ranked with highest score is the most stable.

Preferably, the immunoreactivity is determined using a homogeneous immunoassay concept or a heterogenous immunoassay, such as, ELISA.

A second aspect of the invention relates to the use of a method as defined in the first aspect in a microtiter plate or in a microfluidic format.

Homogeneous immunoassays that do not require a washing step hold particular promise for succesful application in ultra high throughtput screenings methodologies, like, microdroplet- based applications, where solid-phase immobilization and wash steps would be quite challenging.

Formats better suited for high throughput screening have been developed in recent decades under various commercial names, such as: Alpha, AlphaScreen, AlphaLisa, LANCE and SPARCL (non-exhaustive list).

EXAMPLES

Materials and Methods

Composition of model detergent A (liquid)

Ingredients: 12% LAS, 1 1 % AEO Biosoft N25-7 (Nl), 7% AEOS (SLES), 6% MPG

(monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soya soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1 % sodium formiate, 0.2% DTMPA and 0.2% PCA (all percentages are w/w). Example 1. Activity vs. immunoassay of parent and variant amylases

Immunoassays and, especially, ELISA-based assays have been very well-known for decades; a general description is provided, for example, by Wikipedia.

Herein, a purified Parent amylase (amino acid sequence shown in SEQ ID NO:1 ) and a purified variant thereof containing a single amino acid substitution, V206Y, were diluted in a 500 mM HEPES pH7,5; 10 mM EDTA, 0,01 % Tween-20 buffer and temperature-stressed in a PCR- instrument at 55°C over different periods of time to compare the stability in terms of residual amylase activity. The samples were stressed for different periods of time, removed, cooled and analyzed by two different methods:

The samples were analyzed by a standard enzymatic activity assay (G7 amyl-substrate). The activities were transformed into a concentration measure - using a standard curve of the parent amylase.

Percent protein (amylase) remaining after stress (PPRAS) was calculated: (Concentration at time t)/(Concentration at time t=0)^*100%

In parallel, the immunoreactivity of the samples was measured by an immunoassay (ELISA). The immunoreactivity signals were transformed into an "Intact protein equivalents" (IPE) score - using a standard curve of the intact protein (parent amylase).

Percent protein (amylase) remaining after stress (PPRAS) was calculated: (IPE at time t)/(IPE at t=0)^*100%

Enzymatic assay:

AMYL 1 1876473 - 316 Kit (Roche/Hitachi - Roche diagnostics, Mannheim) contains a synthetic amylase substrate, which after being cleaved by the amylase, is further processed by an amyloglucosidase in the kit, leading to release of para-nitro-phenol (pNP), which is then measured spectrophotometrically (OD405 nm) in a microtiterplate (MTP) reader. Immunoassay (exemplified by ELISA):

ELISA immunoassay was carried out in a blocked 384-well MTP coated with "protein A"- purified rabbit Polyclonal-anti-"Parent amylase" antibodies. Samples/standards/controls were added to the washed blocked coated wells followed by incubation. Detection was carried out using the same polyclonal antibodies, however in a horseradish peroxidase-labeled form (LL-HRP conjugation). The immunoassay signal was read as end-point OD-(620 nM) subsequent to substrate addition.

Procedure: Coating: Anti-"parent amylase" antibody in PBS overnight @ +4°C. Wash 3 times and block with TBS-T buffer. Wash 3 times and add samples and standards diluted in TBS- T. Wash 3 times and add anti-parent amylase:HRP conjugate (appropriate dilution) in TBS-T. Wash 3 times and add substrate (TMB-plus). Measure: OD-620 end-point. All incubation: 20 min.

Materials and reagents: MaxiSorp plate, Nunc 384 MTP (Assay plate); Protein A purification kit, Sigma PURE 1A; LL-HRP conjugation kit, NovusBio, cat. no: 701 -0000; TMB-Plus substrate, KemEnTec, cat. no: 4390A; parent amylase antibody; Precipitated/dialyzed and sterile filtered parent amylase; LL-HRP conjugated Protein A purified anti-parent amylase antibody, in stabilizing buffer from Pierce. TBS-T - buffer: 0.15M NaCI; 0,02M Tris; pH7,5; 0,1 % Tween-20 (store @ +4°C); PBS pH7,2 - buffer: 0,137M NaCI; 0,003M KCI; 0,002M KH2P04; 0,005M K2HP04 (store @ +4°C) Enzymatic assay Immunoassay (ELISA)

Incubation- Parent amylase Parent amylase

time (min) Sample-1 Sample-2 Sample-1 Sample-2

15 min 45,1 43,7 56,6 45,1

30 min 22,1 20,5 26,6 26,7

60 min 4,0 4,2 4,6 4,4

120 min 0,2 0,0 0,0 0,0

240 min 0 0 0 0

360 min 0 0 0 0

Table 1 . Percent protein (amylase) remaining after stress (PPRAS), measured in duplicates, after stress @ 55 deg. C. for the parent amylase protein, as a function of time.

2 different methods or measuring stability (PPRAS) are compared.

Table 2. Percent protein (amylase) remaining after stress (PPRAS), measured in duplicates, after stress @ 55 deg. C. for the V206Y amylase variant protein, as a function of time.

2 different methods or measuring stability (PPRAS) are compared. The two different methods (enzymatic assay and immunoassay) give comparable stability results for the parent amylase protein. They also give comparable results for the V206Y variant.

Both assays find the V206Y variant stabilized relative to parent, since degradation is slower for the variant. Example 2. Activity vs. immunoassay of parent and variant pectate lyase

The degradation of a parent pectate lyase (amino acid sequnce shown in SEQ ID NO:2) follows the expected exponential decay when monitored using an immunoassay of the invention: Both at 52 and 55 degrees Celcius; as shown in table 3 and table 4. The degradation of a stabilized variant of the parent pectate lyase denoted Variant #4, which contains the single amino acid substitution: T49R, also follows the expected exponential decay at 55 degrees Celcius, when monitored using ELISA, as shown in table 5. Pectate lyase samples were tested at 3-4 different initial/start-concentrations (3-fold dilution factor between them).

They were all diluted 1 :9 with Model A detergent. Detergent diluted samples were stressed at elevated temperature for a period of time in a PCR instrument. Stressed (and unstressed samples (incubation time = 0)) were diluted 10 fold in ELISA assay buffer (TBS-T buffer) and immunoreactivity measured by ELISA.

Enzyme concentrations found in the temperature-stressed samples are reported in tables 3, 4 and 5 as intact protein equivalents (IPE).

The theoretical degradation curves were calculated by assuming exponential decay using the formula: Cone (t) = Cone. (t=0) ^* (½)^A(t/t½).

Table 3: Half-life ( t½ ) = 52 min; table 4: Half-life ( t½ ) = 13 min and table 5: Half-life ( t½ )

= 26 min.

Immunoassay:

ELISA quantification was carried out in blocked 384-well MTP coated with "protein A"- purified rabbit polyclonal anti-parent pectate lyase antibodies. Samples/standards/controls were added to the washed blocked coated wells followed by incubation. Detection was carried out using the same polyclonal antibodies, however in a HRP-labeled form (LL-HRP conjugation). The immunoassay signal was read as end-point OD-(620 nM) subsequent to substrate addition.

Procedure: Coating: Anti-parent pectate lyase antibody in PBS O/N @ +4°C. Wash 3 times and block with TBS-T buffer. Wash 3 times and add samples and standards diluted in TBS- T. Wash 3 times and add anti-parent pectate lyase:HRP conjugate (appropriate dilution) in TBS- T. Wash 3 times and add substrate (TMB-plus). Measure: OD-620 end-point. All incubation: 20 min.

Materials and reagents: MaxiSorp plate, Nunc 384mtp (Assay plate); Protein A purification kit, Sigma PURE 1A; LL-HRP conjugation kit, NovusBio, cat. no: 701 -0000; TMB-Plus substrate, KemEnTec, cat. no: 4390A; parent pectate lyase antibody; precipitated/dialyzed and sterile filtered Parent pectate lyase; LL-HRP conjugated Protein A purified anti-parent pectate lyase antibody, in stabilizing buffer from Pierce. TBS-T - buffer: 0,15M NaCI; 0,02M Tris; pH7,5; 0,1 % Tween-20 (store @ +4°C); PBS pH7,2 - buffer: 0,137M NaCI; 0,003M KCI; 0,002M KH2P04; 0,005M K2HP04 (store @ +4°C) Incubation temp (°C) 5 52 52 52 52 52

Start-conc. Incubation-time (min) 0 30 60 90 120 180

X ELISA: 0,826 nd nd nd 0,132 0,100

X Theoretical decay: 0,826 0,554 0,371 0,249 0,167 0,075

ELISA: 0,251 0,165 0,124 0,089 0,060 0,034

X/3 Theoretical decay: 0,275 0,185 0,124 0,083 0,056 0,025

ELISA: 0,096 0,053 0,041 0,029 0,023 0,014

X/9 Theoretical decay: 0,092 0,062 0,041 0,028 0,019 0,008

ELISA: nd 0,020 0,014 0,01 1 nd nd

X/27 Theoretical decay: 0,031 0,021 0,014 0,009 0,006 0,003

Table 3: Strength (Intact protein equivalents (IPE)) measured by ELISA as a function of incubationtime and start-conc is shown. Also the theoretical values are shown.

Degradation of parent pectate lyase @ 52°C, follows the theoretical decay (T½ = 52 min.) when measured by ELISA. (nd =no data (The actual diluted sample tested, was outside the dynamic range of the ELISA assay))

Incubation-temp (°C) 5 55 55 55 55 55

Start-conc. Incubation-time (min) 0 30 60 90 120 180

X ELISA: 0,826 0,140 0,054 0,016 nd nd

X Theoretical decay: 0,826 0,167 0,034 0,007 0,001 0,000

ELISA: 0,251 0,051 0,019 nd nd nd

X/3 Theoretical decay: 0,275 0,056 0,01 1 0,002 0,000 0,000

ELISA: 0,096 0,018 nd nd nd nd

X/9 Theoretical decay: 0,092 0,019 0,004 0,001 0,000 0,000 Table 4: Strength (Intact protein equivalents (IPE)) measured by ELISA as a function of incubationtime and start-conc is shown. Also the theoretical values are shown. Degradation of parent pectate lyase @ 55°C, follows the theoretical decay (T½ = 13 min) when measured by ELISA. (nd =no data (The actual diluted sample tested was outside the dynamic range of the ELISA assay)) Incubation-temp (°C) 5 55 55 55 55 55

Start - cone. Incubation-time (min) 0 30 60 90 120 180

Y ELISA: 0,347 0,125 0,070 0,042 0,021 nd

Y Theoretical decay: 0,347 0,156 0,070 0,032 0,014 0,003

ELISA: 0,124 0,047 0,026 0,016 nd nd

Y/3 Theoretical decay: 0,124 0,056 0,025 0,01 1 0,005 0,001

ELISA: nd 0,018 nd nd nd nd

Y/9 Theoretical decay: 0,041 0,019 0,008 0,004 0,002 0,000

Table 5: Strength (Intact protein equivalents (IPE)) measured by ELISA as a function of incubationtime and start-cone is shown. Also the theoretical values are shown. Degradation of pectate lyase variant (Var. #4) @ 55°C, follows the theoretical decay (T½ = 26 min) when measured by ELISA. (nd =no data (The actual diluted sample tested, was outside the dynamic range of the ELISA assay))

Monitoring the degradation of a protein as a function of time using an immunoassay give results in accordance with the theoretically expected pattern.

This is also true for a variant of the same enzyme - even though the same polyclonal antibody is used.

Example 3. Activity vs. immunoassay of parent and variant pectate lyases

Pectate lyase variants were stressed in a liquid detergent at elevated temperature.

Samples were removed as a function of time and percent protein remaining after stress (PPRAS) measured by means of an enzyme activity assay and an immunoassay (ELISA).

The parent pectate lyase and 8 variants thereof were tested; the parent and one variant

(Var. #8) were tested in duplicate.

The 8 variants are protein engineered variants of the parent Pectate Lyase with the following substitutions:

Var. #1 : E108N; Var. #2: T49W K99D E108N S176D I325F Q356F; Var. #3 K99D S176D I325F Q356F; Var. #4: T49R; Var. #5: I250L; Var. #6: I250N; Var. #7: S229I; Var. #8:T49R K99D S176D S229I K257L I325F Q356F.

Half-life improvement factors (HIF) were calculated in both assays and compared. The

HIF factors correlate well (shown in table 6), demonstrating that an immunoassay is suited for indirectly evaluating the stability of protein engineered variants. Stress testing:

9 purified samples were prepared: The parent pectate lyase and variants #1 - #8 (see list of variants). 1000 ppm sample mixed 1 :9 with Persil Small&Mighty non-bio.

The mixtures were transferred individually to separate PCR-tubes and stressed at 55°C in PCR-machine (PTC200), for the listed periods of time: 0, 60, 120, 240 and 960 min, enabling characterization of degradation curves and calculation of T½-values.

Pectate lyase activity assay:

Based on published principle: Collmer et al.; Methods in enzymology 1988, Vol. 161.

Materials: Spectra plates 384well, Perkin Elmer #6007649; Buffer: 100mM Tris/HCI, 0,68mM CaCI2, pH8,0; Polygalacturonic Acid, Sigma P3850; UV plate, 384well, Greiner #781801 - for OD-measurement only; Spectrophotometer, Tecan (kinetic meas. at 235nm).

Stressed samples were diluted 250 fold in Buffer and then mixed 1 :1 with polygalacturonic acid substrate (2300 g. Supernatant from spun 1 % stirred solution) in 384-well optical clear MTPs. The MTPs were measured kinetically at OD235nm once every minute for 30 min. The pectate lyase cone. Was calculated from a Parent pectate lyase standard-curve.

Pectate Lyase ELISA:

Samples diluted 250-fold for enzymatic activity measurement were further diluted F=20 in ELISA assay buffer (TBS-T). Standard sandwich ELISA set-up in 384-well plates (see example 2), based on polyclonal rabbit anti-parent pectate lyase antibodies. Detect-antibody was HRP- conjugated. IPE measured using ELISA.

Calculations:

The Excel "Logest" Function calculates the exponential curve that best fits a supplied set of y- and x- values, y = b * m^Ax . From here we can calculate the exponential decay constant: (k=Ln(m)) and the T½ time (T½=Ln2/k). Where X is incubation time and Y is PPRAS.

The HIF factor for a variant is calculated as variant half-life divided by parent half-life (T½(var.) / T½(parent). As shown in table 6 below, the variants score similar half-life improvement factors (HIF) in both assays:

Var. #4 3,8 2,3

Var. #5 6,0 6,8

Var. #6 8,1 8,0

Var. #7 9,6 8,9

Var. #8 rep-1 >15 >15

Var. #8 rep-2 >15 >15

Table 6: HIF values listed for a number of variants measured by two different methods. Stability for a number of pectate lyase variants and their parent measured in 2 different assays: An enzymatic activity assay (Pec. Ly. Activity) and an immunoassay (ELISA). Comparable HIF factors values were determined in the two assays.

Claims

1 . A method for determining the stability of at least one biologically active polypeptide of interest, said method comprising the steps of:

a) providing the at least one biologically active polypeptide;

2. The method of claim 1 , wherein the at least one biologically active polypeptide is one or more enzyme, preferably selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectate lyase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase.

3. The method of any preceding claim, wherein the at least one biologically active polypeptide consists of a single domain; preferably the at least one biologically active polypeptide consists of at most 1 ,000 amino acids; preferably at most 950 amino acids; more preferably at most 900, 850, 800, 750, 700, 650, 600, 550, or most preferably at most 500 amino acids.

4. The method of any preceding claim, wherein the at least one biologically active polypeptide comprises a parent polypeptide and one or more variant thereof, wherein the one or more variant comprises at least one amino acid alteration, such as, a deletion, insertion or substitution of at least one amino acid in one or more positions, and wherein the one or more variant comprises an amino acid sequence at least 70% identical to that of the parent; preferably at least 75% identical, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of the parent.

5. The method of claim 4, comprising an additional step of identifying and selecting at least one variant having a higher or lower stability over said period of time than its parent.

6. The method of any preceding claim, wherein the one or more conditions of interest comprise temperature, pH, surfactant concentration, salt concentration, Ca++ concentration, and/or EDTA concentration.

7. The method of any of preceding claim, wherein the at least one biologically active polypeptide of interest is comprised in one or more composition; preferably said composition is a laundry detergent composition, an automatic dishwasher detergent composition, a food processing aid composition, a food additive composition, a beverage processing aid composition, a beverage additive composition, a feed processing aid composition, a feed additive composition, a seed-coating composition, an agricultural inoculant composition or an industrial enzyme composition.

8. The method of any preceding claim which is carried out in a microtiterplate format.

9. The method of any of claims 1 - 7 which is carried out in a microfluidic format, wherein the sample of step (a) is comprised in an aqueous microdroplet in an immiscible carrier fluid.

10. The method of any preceding claim, wherein the immunoreactivity of the at least one biologically active polypeptide of interest is determined in step (c) by comparison with an immunoreactivity standard curve of a reference biologically active polypeptide of interest; preferably the at least one biologically active polypeptide is a variant of a parent polypeptide which is the reference.

1 1 . The method of any preceding claim, wherein the immunoreactivity is determined using an antibody raised against the purified biologically active polypeptide of interest; preferably the antibody is polyclonal; even more preferably the polyclonal antibody is raised in rabbit.

12. The method of any preceding claim, wherein steps (a) through (c) are repeated at least once, wherein the immunoreactivity determinations are compared and the polypeptides are ranked, with the polypeptide having the smallest percentage difference between the immunoreactivity measurements as the most stable.

13. The method of claim 12, wherein immunoreactivity determinations are converted into Intact Polypeptide Equivalents, IPE, using an immunoreactivity standard curve constructed using the parent polypeptide or a variant thereof, wherein %-residual IPE is calculated for each biologically active polypeptide of interest, and wherein the biologically active polypeptide of interest which is ranked with highest score is the most stable.

14. The method of any preceding claim, wherein the immunoreactivity is determined using a homogeneous immunoassay concept or a heterogenous immunoassay, such as, ELISA.

15. Use of a method as defined in any preceding claim in a microtiter plate or in a microfluidic format.