Cation chromatography using predicted elution buffer salt concentration

1 . A chromatography method of producing a target elution volume comprising a first and a second target protein, the method comprising:

providing a cation exchange chromatography column;

applying a protein solution on the column, the protein solution comprising at least the first target protein and the second target protein and optionally one or more further proteins;

inputting an optimization criterion into a chromatography simulation software, the optimization criterion being a desired property of the target elution volume in respect to the first and second target proteins comprised in the target elution volume;

computing, by the chromatography simulation software, an elution buffer salt concentration adapted to elute the first and the second target proteins from the chromatography column such that a target elution volume can be obtained that matches the optimization criterion best, the computation comprising computing a plurality of chromatography simulations as a function of multiple different elution buffer salt concentrations;

computing, by the chromatography simulation software, pooling borders of the target elution volume as a function of at least the computed salt concentration and the input optimization criterion;

applying an elution buffer having the computed salt concentration on the chromatography column;

performing the elution using the applied elution buffer; and

collecting the computed target elution volume as a separate fraction using the computed pooling borders,

wherein the first and the second target protein are different glycoforms of a monomeric Immunoglobulin G (IgG) Fc-fusion protein of a brain shuttle molecule linked to an immunoglobulin,

wherein the brain shuttle molecule a molecule that is able to increase penetration of large molecules such as antibodies into the brain.

2 . The chromatography method of claim 1 , the optimization criterion being selected from a group comprising:

a) a desired ratio or ratio range of the amounts of the first and of the second target proteins in the target elution volume;

b) a desired amount or amount range of the first target protein in combination with a desired amount or amount range of the second target protein in the target elution volume;

c) a desired purity or purity range of the first target protein in combination with:

a desired ratio of the amounts of the first and second protein in the target elution volume

a desired amount or amount range of the second target protein in the target elution volume;

a desired purity or purity range of the second target protein in the target elution volume; and

d) a combination of two or more of the optimization criterion.

3 . The chromatography method of claim 1 ,

wherein the applied protein solution comprises the one or more further proteins.

4 . The chromatography method of claim 1 ,

wherein the plurality of chromatography simulations are computed as a function of the multiple different elution buffer salt concentrations and as a function of multiple different elution buffer pH values, the method further comprising:

wherein the computing comprises computing a combination of an elution buffer salt concentration and an elution buffer pH value which in combination are adapted to elute the first and the second target proteins from the chromatography column such that a target elution volume can be obtained that matches the optimization criterion best, wherein the chromatography simulations are performed for identifying the combination of the combination of the elution buffer salt concentration and the elution buffer pH;

wherein the pooling borders of the target elution volume are computed as a function of at least the computed combination of the elution buffer salt concentration and the elution buffer pH value and the input optimization criterion; and

wherein the elution buffer that is applied on the column has the computed salt concentration and the computed pH value.

5 . The method of claim 1 , wherein the first and the second target proteins are proteins having a resolution factor of less than 0.75.

6 . The method of claim 1 , wherein the first and the second target proteins are glycosylation variants of proteins having an identical amino acid sequence.

7 . The method of claim 1 , wherein the first and the second target proteins are antibody monomers having an identical amino acid sequence and comprising different numbers of glycosyl groups on the FAB fragment.

8 . The method of claim 1 , wherein the applied protein solution comprises each of the target proteins and each of the further proteins, if any, in a respective concentration of at least 0.5% by weight.

9 . The method of claim 1 , wherein the second target protein and one or more of the further proteins, if any, comprised in the applied protein solution have an affinity to the stationary phase of the column that is similar as the affinity of the first target protein to the stationary phase leading to overlapping elution behaviors.

10 . The method of claim 1 , wherein a total amount of protein in the protein solution applied to the column is identical to or smaller than the maximum protein load capacity of the column, and is in a range of 50% to 100% of the maximum protein load capacity.

11 . The method of claim 1 , wherein the computed pooling borders of the target elution volume are specified in a form of a collection start time offset and a collection stop time offset, the method comprising:

continuously monitoring, by an automated chromatography system comprising the chromatography column, the time lapsed since the starting of the elution;

automatically starting the collecting of the eluted elution buffer by the chromatography system when the lapsed time equals the collection start time offset; and

stopping the collecting of the eluted elution buffer by the chromatography system when the lapsed time equals the collection stop time offset.

12 . The chromatography method of claim 1 ,

wherein each of the plurality of chromatography simulations is a simulation of a chromatography process using two or more elution steps, whereby in each elution step, an elution buffer with a different elution salt concentration is used,

wherein the computed elution buffer salt concentration is a series of different, elusion-step specific elution buffer salt concentrations, and

wherein the applying of the elution buffer having the computed salt concentration on the chromatography column comprises step-wise applying a series of elution buffers having the different salt concentrations in accordance with the computed series of elusion-step specific salt concentrations.

13 . The chromatography method of claim 1 , the method comprising:

inputting an amount of each of the first and second target proteins comprised in the applied protein solution and optionally also the amount of each of one or more further proteins comprised in the applied protein solution, if any, into the chromatography simulation software;

the simulations being computed as a function of a set of parameter values comprising at least:

the dimension of the provided cation exchange chromatography column; and

the amounts of the first and second target proteins and optionally also the amounts of the one or more further proteins applied on the column.

14 . The chromatography method of claim 13 , wherein the set of parameters further comprises:

a predefined pH value of the elution buffer,

a flow rate of the elution buffer through the column; and

chemical properties of the proteins of the applied protein solution.

15 . The method of claim 1 , wherein the chromatography simulation software is configured to use a combination of mathematical models for computing the simulations and/or for computing the pooling borders of the target elution volume, the models comprising:

a column model being configured to interrelate the concentration of each of the proteins, the salt concentration and the pH-value in the elution buffer in the interstitial volume of the column; and

a pore model being configured to interrelate the concentration of each of the proteins, the salt concentration and the pH value in the elution buffer in the pore volume of the stationary phase of the column; and

a reaction model being configured to interrelate the concentration of each of the proteins in the stationary phase, the elution buffer salt concentration and at least some of the chemical properties of each of the proteins in the protein solution.

16 . The method of claim 1 , wherein the first and the second target protein being different glycoforms of a monomeric Immunoglobulin G (IgG) Fc-fusion protein of a brain shuttle molecule linked to an immunoglobulin, wherein the brain shuttle molecule is a molecule that is able to increase penetration of large molecules such as antibodies into the brain, wherein the fusion protein comprises:

i. an antibody consisting of a full length antibody comprising two pairs each of a full length antibody light chain and a full length antibody heavy chain, and

ii. an additional Fab fragment.

17 . The method of claim 16 , wherein the additional Fab-fragment is the brain shuttle molecule linked to the immunoglobulin.

18 . The method of claim 1 , wherein the first and the second target protein being different glycoforms of a monomeric Immunoglobulin G (IgG) Fc-fusion protein of a brain shuttle molecule linked to the immunoglobulin, wherein the fusion protein is a bispecific antibody composed of four polypeptides comprising:

one full length antibody comprising two pairs each of a full length antibody light chain and a full length antibody heavy chain, wherein the light chain has a sequence identity to SEQ ID NO: 01 of 70% or more, wherein the heavy chain has a sequence identity to SEQ ID NO: 02 of 70% or more, wherein the binding sites formed by each of the pairs of the full length heavy chain and the full length light chain specifically bind to a first antigen, and

one additional Fab fragment having a light chain that has a sequence identity to SEQ ID NO: 03 of 70% or more, and a heavy chain Fab fragment that has a sequence identity to SEQ ID NO: 04 of 70% or more, wherein the additional Fab fragment is fused to the C-terminus of one heavy chain of the full length antibody.

19 . The method of claim 18 , wherein the light chains of the full length antibody have the amino acid sequence of SEQ ID NO: 01, the first heavy chain of the full length antibody has the amino acid sequence of SEQ ID NO: 02, the light chain of the Fab fragment has the amino acid sequence of SEQ ID NO: 03, and the second heavy chain of the full length antibody fused to the heavy chain of the Fab fragment has the amino acid sequences of SEQ ID NO: 05.

20 . A chromatography control system comprising a simulation software, the simulation software being configured for performing a method of obtaining pooling borders of a target elution volume comprising a first and a second target protein, the chromatography control system being configured for:

receiving an optimization criterion and inputting the optimization criterion into the chromatography simulation software, the optimization criterion being a desired property of the target elution volume in respect to the first and second target proteins comprised in the target elution volume;

computing, using the chromatography simulation software, an elution buffer salt concentration adapted to elute the first and the second target proteins from the chromatography column such that a target elution volume can be obtained that matches the optimization criterion best, the computing comprising computing a plurality of chromatography simulations as a function of multiple different elution buffer salt concentrations;

computing the pooling borders of the target elution volume as a function of at least the computed salt concentration and the input optimization criterion; and

outputting the computed salt concentration and pooling borders,

wherein the first and the second target protein being different glycoforms of a monomeric Immunoglobulin G (IgG) Fc-fusion protein of a brain shuttle molecule linked to an immunoglobulin, and

wherein the brain shuttle molecule is a molecule that is able to increase penetration of large molecules such as antibodies into the brain.

21 . The chromatography control system of claim 20 , the system further being configured for:

receiving dimensions of a cation exchange chromatography column;

receiving the amounts of the first and second target proteins and optionally also the amounts of the one or more further proteins applied on the column;

wherein the simulations and/or the pooling borders are computed as a function of a set of parameter values comprising at least:

the dimension of the provided cation exchange chromatography column; and

the amounts of the first and second target proteins and optionally also the amounts of the one or more further proteins applied on the column.

22 . The chromatography control system of claim 20 ,

wherein the plurality of chromatography simulations are computed as a function of the multiple different elution buffer salt concentrations and as a function of multiple different elution buffer pH values, the method further comprising:

wherein the computing comprises computing a combination of an elution buffer salt concentration and an elution buffer pH value which in combination are adapted to elute the first and the second target proteins from the chromatography column such that a target elution volume can be obtained that matches the optimization criterion best;

wherein the computing of the pooling borders of the target elution volume is computed as a function of at least the computed combination of the elution buffer salt concentration and the elution buffer pH value and the input optimization criterion; and

wherein the computed pH value is output in addition to the computed elution buffer.

23 . The chromatography control system of claim 22 , wherein

the chromatography control system is configured to control a buffer mixing unit as to automatically generate an elution buffer having the output elution salt concentration; and/or

the chromatography control system is configured to control a buffer mixing unit as to automatically generate an elution buffer having both the salt concentration and the pH value computed in combination and output according to claim 18 ; and/or

the chromatography control system is configured to control an elution buffer selection unit adapted to automatically select one out of a plurality of available elution buffers having different salt concentrations, the selected elution buffer having the output salt concentration; and/or

the chromatography control system is configured to control an elution buffer selection unit adapted to automatically select one out of a plurality of available elution buffers having different salt concentrations and different pH values, the selected elution buffer having both the salt concentration and pH value computed in combination and output according to claim 18 ; and/or

the chromatography control system is configured to control a buffer application unit configured to automatically apply an automatically generated or selected elution buffer on the chromatography column, the applied elution buffer having the output salt concentration or having both the salt concentration and pH value computed in combination and output according to claim 18 ; or

the chromatography control system is configured to control an elution volume collection unit of a chromatography system such that the computed target elution volume is automatically collected as a separate fraction in accordance with the computed pooling borders.

24 . A chromatography system comprising the chromatography control system of claim 23 , and further comprising the buffer mixing unit and/or the elution buffer selection unit and/or the buffer application unit and/or the automated elution volume collection unit.

25 . A non-transitory computer readable medium storing a computer program, which when executed by a computer system, causes the computer system perform a method of managing a chromatography process such that a target elution volume comprising a first and a second target protein is obtained, the computer program comprising a chromatography simulation software and the computer system further configures to perform

receiving an optimization criterion being a desired property of a target elution volume in respect to a first and second target proteins comprised in the target elution volume;

computing, using the chromatography simulation software, an elution buffer salt concentration adapted to elute the first and the second target proteins from the chromatography column such that a target elution volume can be obtained that matches the optimization criterion best, the computing comprising computing a plurality of chromatography simulations as a function of multiple different elution buffer salt concentrations;

computing the pooling borders of the target elution volume as a function of at least the computed salt concentration and the input optimization criterion; and

outputting the computed salt concentration and/or the pooling borders for enabling a user to control a chromatography system such that a target elution volume comprising the first and the second target proteins in accordance with the optimization criterion is obtained and/or using the computed salt concentration and/or the pooling borders for automatically or semi-automatically controlling a chromatography system such that a target elution volume comprising the first and the second target proteins in accordance with the optimization criterion is obtained,

wherein the first and the second target protein being different glycoforms of a monomeric Immunoglobulin G (IgG) Fc-fusion protein of a brain shuttle molecule linked to an immunoglobulin,

wherein the brain shuttle molecule is a molecule that is able to increase penetration of large molecules such as antibodies into the brain.

26 . The non-transitory computer-readable medium of claim 25 , wherein the computer system is further configured for generating control commands for automatically or semi-automatically controlling one or more units of a chromatography system.