METHOD OF SYNTHESIZING ANTIBODY DRUG CONJUGATES USING AFFINITY RESINS
20170326251 · 2017-11-16
Inventors
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
A61K47/6811
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
International classification
A61K47/68
HUMAN NECESSITIES
Abstract
Disclosed is a solid phase method of synthesizing biomolecule-drug-conjugates. In particular, this invention relates to a solid phase method of synthesizing antibody-drug-conjugates (ADCs). This invention also relates to intermediate methods of producing immobilized, chemically modified biomolecules, e.g., antibodies.
Claims
1. A method of synthesising synthesizing a biomolecule-drug-conjugate, the method comprising: a) optionally contacting a biomolecule with an agent selected from the group consisting of a chemical modification agent, an enzymatic modification agent, and an activating agent to provide a chemically modified, enzymatically modified, or activated biomolecule; b) (i) when step (a) is carried out, contacting the chemically modified, enzymatically modified, or activated biomolecule of step (a) with a capture resin comprising a non-peptide based capture moiety selected from the group consisting of Protein A, Protein G and Protein L mimetic biomolecule capture moiety under conditions suitable to immobilise the chemically modified, enzymatically modified, or activated biomolecule and therefore provide an immobilised chemically modified, enzymatically modified, or activated biomolecule; or (ii) when step (a) is not carried out, contacting a biomolecule with a capture resin comprising a non-peptide based capture moiety selected from the group consisting of Protein A, Protein G and Protein L mimetic biomolecule capture moiety under conditions suitable to immobilise the biomolecule and therefore provide an immobilised biomolecule; c) optionally contacting the immobilised chemically modified, enzymatically modified, or activated biomolecule of step (b) (i) or the immobilised biomolecule of step (b) (ii) with an agent selected from the group consisting of a chemical modification agent, an enzymatic modification agent, and an activating agent to provide an immobilised chemically modified, enzymatically modified, or and/or activated biomolecule; d) optionally washing the immobilised chemically modified, enzymatically modified, or activated biomolecule of step (b) (i); the immobilised biomolecule of step (b) (ii); or the immobilised chemically modified, enzymatically modified, or activated, immobilised biomolecule of step (c) with buffer to remove superfluous or unreacted chemical modification agent, enzymatic modification agent, or superfluous or unreacted activating agent, e) optionally repeating step (c) and step (d); f) optionally contacting a drug component with an agent selected from the group consisting of a chemical modification agent, an enzymatic modification agent, and an activating agent to provide a chemically modified, enzymatically modified, or activated drug component; g) (i) when step (f) is carried out, contacting the immobilised biomolecule or the immobilised chemically modified, enzymatically modified, or and/or activated biomolecule with the chemically modified, enzymatically modified, or activated drug component of step (f) to form an immobilised biomolecule-drug-conjugate; or (ii) when step (f) is not carried out contacting the immobilised biomolecule or the immobilised chemically modified, enzymatically modified, or and/or activated biomolecule with a drug component to form an immobilised biomolecule-drug-conjugate; h) optionally washing the immobilised biomolecule-drug-conjugate of step (g) with buffer to remove superfluous or unreacted reagents, to provide a purified immobilised biomolecule-drug conjugate; i) releasing the purified biomolecule-drug-conjugate from the capture resin; wherein the biomolecule is selected from the group consisting of an antibody, a modified antibody, and an antibody fragment.
2. (canceled)
3. The method of claim 1, wherein step (a) is carried out.
4. The method of claim 3, wherein step (a) involves reducing the biomolecule.
5. The method of claim 3, wherein step (a) involves reacting the biomolecule with a crosslinker moiety, optionally wherein the crosslinker moiety is an amine-to-sulfhydryl crosslinker.
6. The method of claim 5, wherein the crosslinker moiety is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
7. The method of claim 1 any preceding claim, wherein step (b) involves incubation with the capture resin, optionally wherein the incubation is carried out at temperature of from about 5° C. to about 50° C. or and/or optionally wherein the incubation is for a period of time of from about 10 minutes to about 18 hours.
8. (canceled)
9. The method of claim 1, wherein step (c) is carried out.
10. The method of claim 9, wherein step (c) involves reducing the biomolecule.
11. The method of claim 9, wherein step (c) involves reacting the biomolecule with a crosslinker moiety, optionally wherein the crosslinker moiety is an amine-to-sulfhydryl crosslinker.
12. The method of claim 11, wherein the crosslinker moiety is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
13. The method of claim 1, wherein step (d) is omitted.
14. The method of claim 1, wherein step (d) is carried out, optionally wherein the washing involves rinsing with a buffer, further optionally wherein the buffer is phosphate buffered saline (PBS).
15. (canceled)
16. The method of claim 1, wherein step (f) is carried out.
17. The method of claim 16, wherein the step of contacting the drug component with a chemical modification agent, an enzymatic modification agent, or an activating agent to provide a modified or activated drug component involves reacting the drug component with a crosslinker moiety, optionally wherein the crosslinker moiety is an amine-to-sulfhydryl crosslinker.
18. The method of claim 17, wherein the crosslinker moiety is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
19. The method of claim 1, wherein step (g) involves simultaneously (1) carrying out the chemical modification, enzymatic modification or activation of the drug component and (2) contacting the immobilised biomolecule or the chemically modified, enzymatically modified, or and/or activated, immobilised biomolecule.
20-21. (canceled)
22. The method of claim 1, wherein step (h) is carried out, optionally wherein the washing involves rinsing with a buffer, further optionally wherein the buffer is phosphate buffered saline (PBS).
23. The method of claim 1, wherein step (i) involves a) exposing the support-biomolecule compound to a release agent; or b) altering the pH to break the support-biomolecule bond.
24. The method of claim 1, wherein the capture resin is selected from the group consisting of Fabsorbent™ F1P HF resin, Mabsorbent™ A1P resin, and Mabsorben™ A2P resin.
25. A method of synthesizing a chemically modified, an enzymatically modified, or an activated, immobilised biomolecule, the method comprising: (a) optionally contacting a biomolecule with an agent selected from the group consisting of a chemical modification agent, an enzymatic modification agent, and an activating agent to provide a chemically modified, enzymatically modified, or activated biomolecule; (b) (i) when step (a) is carried out, contacting the chemically modified, enzymatically modified, or activated biomolecule of step (a) with a capture resin comprising a non-peptide based capture moiety selected from the group consisting of Protein A, Protein G, Protein L mimetic biomolecule capture moiety under conditions suitable to immobilise the chemically modified, enzymatically modified, or activated biomolecule and therefore provide an immobilised chemically modified, enzymatically modified or activated biomolecule; or (ii) when step (a) is not carried out, contacting a biomolecule with a capture resin comprising a non-peptide based capture moiety selected from the group consisting of Protein A, Protein G, and Protein L mimetic biomolecule capture moiety under conditions suitable to immobilise the biomolecule and therefore provide an immobilised biomolecule; (c) contacting the immobilised chemically modified, enzymatically modified, or activated biomolecule of step (b) (i); or the immobilised biomolecule of step (b) (ii) with an agent selected from the group consisting of a chemical modification agent, an enzymatic modification agent, and an activating agent to provide an immobilised chemically modified, enzymatically modified, or activated biomolecule; wherein the biomolecule is selected from the group consisting of an antibody, a modified antibody, and an antibody fragment.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0315] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
[0316]
[0317]
[0318]
[0319]
[0320]
[0321] The chromatographic data demonstrates that increasing the TCEP to Antibody ratio increases the average drug antibody ratio (DAR) and that as DAR increases there is no decrease in monomer content using the solid phase technique.
[0322]
[0323]
[0324]
DETAILED DESCRIPTION
[0325] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0326] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0327] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
EXAMPLES
[0328] The following techniques are used in the examples.
Size Exclusion Chromatography (SEC)
[0329] Size exclusion chromatography was performed using a TOSOH Bioscience TSK-Gel® GW3000SWxI column in 0.2M potassium phosphate pH 6.95 with 0.25 mM potassium chloride and 10% IPA at a flow rate of 0.5 ml/min. Aggregation state of the conjugate was determined by integration of eluted peak area absorbance at 280 nm.
Hydrophobic Interaction Chromatography (HIC)
[0330] Hydrophobic interaction chromatography was performed using a TOSOH TSK-Gel® butyl NPR column with a linear gradient of 0-100% buffer A to B over 12 minutes at a flow rate of 0.8 ml/min. Where buffer A is 1.5 M ammonium acetate pH 6.95 with 25 mM sodium phosphate and buffer B is 25 mM sodium phosphate pH 6.95 with 25% IPA. Antibody drug ratio of the conjugate was determined by integration of eluted peak area absorbance at 280 nm.
Reverse Phase Chromatography (RP-PLRP)
[0331] Reverse phase (Polymer Labs PLRP) chromatography was performed using an Agilent PLRP-S PL1912-1502 column with a gradient of 25-95% buffer A to B over 31 minutes at a flow rate of 0.25 ml/min. Where buffer A is Water with 0.05% TFA and buffer B is ACN with 0.04% TFA. Samples were reduced pre injection with 20 mM sodium borate pH 8.4 containing 50 mM DTT at 37° C. for 15 minutes. Antibody drug ratio of the conjugate was determined by integration of eluted peak area absorbance at 280 nm.
Drug to Antibody Ratio by UV Analysis
[0332] For UV analysis the sample was added to a 400 ul quartz cuvette with a path length of 1 cm and the absorbance at 252 nm and 280 nm measured on a Thermo scientific Multiskan GO spectrophotometer. The 252 nm and 280 nm data was used to calculate Drug antibody ratio based on published molar extinction coefficients for Herceptin and DM1 at these wavelengths.
Example 1
Solid Phase Antibody Drug Conjugate Screening
[0333] This example demonstrates that immobilized antibodies can be conjugated to a defined drug loading with a generic process that negates the need for process development. This approach is suitable for adapting to 96 well plate high throughput screening.
[0334] Herceptin (0.5 ml of 1 mg/ml in PBS, pH 7.4) was bound to 100 μl (settled resin volume) of Fabsorbent™ F1P HF resin equilibrated in PBS by mixing the resin slurry and antibody solution gently for 30 minutes. Unbound Herceptin was removed by washing the resin with PBS, 2 mM EDTA and the resin finally re-suspended in 0.5 ml PBS/EDTA.
[0335] The bound Herceptin (Her) was reduced by adding tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) to a final concentration of 2 mM and then incubating the suspension at ambient temperature for 18 hours. The resin was washed with PBS/EDTA to remove unreacted TCEP and then re-suspended in 475 μl PBS/EDTA.
[0336] vcMMAE (vcE), N-ethyl maleimide (NEM) and dimethylacetamide (DMA) were added to achieve final concentrations of 1 mM maleimide (total vcE and NEM) and 5% v/v DMA. The ratio of vcE to NEM was varied 100:0, 75:25, 50:50, 25:75 and 0:100. The reduced antibody was conjugated by incubating the resin suspension at ambient for 60 minutes. The resin was washed sequentially with PBS/EDTA/5% v/v DMA and 0.1M glycine pH 5.0.
[0337] The conjugates were eluted with 0.1M glycine pH 3.0. The eluted conjugates were collected into 2% v/v of 1M tris(hydroxymethyl)aminoethane (TRIS) to neutralise them.
[0338] The neutralised conjugates were then analysed by Size Exclusion Chromatography and Reverse Phase Chromatography (Polymer Labs, PLRP) Chromatography to determine the percentage aggregate and average drug loading.
[0339] The results are summarized in Table 1 below:
TABLE-US-00002 TABLE 1 Mass of Her Bound (mg/ml Drug to Antibody resin) Ratio of vcE:NEM % Aggregate ratio (DAR) 10 100:0 9.72 7.9 10 75:25 4.69 5.7 10 50:50 3.08 4.4 10 25:75 0.80 2.8 10 0:100 0.42 0.0
[0340] The aggregate content of even the highest drug loaded conjugates is acceptable for further evaluation in antigen binding and cell based assays. The sequential washes with PBS/ETDA/5% v/v DMA and then 0.1M glycine pH 5.0 ensure the final conjugates are free from unreacted drug linker, NEM and solvent and do not compromise interpretation of bioassay data. With Fabsorbentυ F1P HF resin this approach is useful for screening panels of monoclonal antibodies as part of lead selection for subsequent antibody drug conjugation development or for producing ADCs direct from tissue culture supernatants containing both intact and Fab fragment antibodies.
Example 2
Solid Phase Partial TCEP Reduction in Batch Mode
[0341] This example shows that immobilized antibodies can be conjugated to a defined drug loading by partial reduction of the interchain disulphide bonds followed by conjugation with vcMMAE and that product quality is enhanced relative to the same conjugates made in solution.
[0342] Herceptin (0.5 ml of 2 mg/ml PBS, pH 7.4) was bound to 100 μl (settled resin volume) of Fabsorbentυ F1P HF resin equilibrated in PBS by mixing the resin slurry and antibody solution gently for 30 minutes. Unbound Herceptin was removed by washing the resin with PBS, 2 mM EDTA and the resin finally re-suspended in 0.5 ml PBS/EDTA.
[0343] The bound Herceptin was reduced by adding tris-(2-carboxyethyl)phosphine hydrochloride to a ratio of 1 to 4 moles of TCEP per mole of Herceptin and then incubating the suspension at ambient temperature for 2 hours.
[0344] vcMMAE and dimethylacetamide (DMA) were added to achieve 2.5 to 10 moles of vcMMAE per mole of Herceptin and 5% v/v DMA and the conjugation allowed to proceed for 30 minutes at ambient. N-Acetyl cysteine (NAC) was added to quench unreacted vcMMAE and allowed to react for 20 minutes before the resin was washed sequentially with PBS/EDTA/5% v/v DMA and 0.1M glycine pH 5.0.
[0345] The conjugates were eluted with 0.1M glycine pH 3.0 and collected into 2% v/v of 1M tris(hydroxymethyl)aminoethane (TRIS) to neutralise them.
[0346] An equivalent series of solution phase conjugates of Herceptin with vcMMAE with matched DAR were produced and analysed to provide a comparison of solid phase and solution phase conjugate quality.
[0347] The eluted conjugates were then analysed by Hydrophobic Interaction Chromatography (
[0348] The results are summarized in Table 2 below:
TABLE-US-00003 TABLE 2 Solution Solid DAR % Aggregate % Aggregate 0 (Herceptin) 0.2 1.3 0.4 0.3 2.4 0.7 0.3 3.4 1.1 0.3 4.4 1.5 0.3
[0349] The data show that on solid supports the relationship between TCEP to antibody ratio and final drug loading is linear. In addition when compared with an equivalent conjugate made in solution the solid phase conjugates show a lower percentage aggregation.
Example 3
Solid Phase Partial TCEP Reduction on Column
[0350] This example shows that immobilized antibody conjugation can be adapted to a chromatographic flow process with excellent reproducibility.
[0351] Herceptin (5 ml of 2 mg/ml PBS, pH 7.4) was bound to a 1 ml column of Fabsorbent™ F1P HF resin (previously equilibrated in PBS) by loading at 120 cm/hr. The bound Herceptin was prepared for reduction by equilibrating the resin with PBS, 2 mM EDTA.
[0352] A micro peristaltic pump was used to create a small volume PBS/EDTA recirculation loop through the column (approximately 200 μL external to the column) to which TCEP was added to give a molar ratio of 2 TCEP per mole of Herceptin. This was allowed to recirculate for 120 minutes at ambient to reduce the Herceptin.
[0353] The contents of the reservoir and column were flushed to waste and replaced with PBS/EDTA/5% v/v DMA to which vcMMAE was added to give a molar ratio of 5 vcMMAE per mole of reduced Herceptin. This was allowed to recirculate for 60 minutes at ambient to conjugate the reduced Herceptin.
[0354] N-Acetyl cysteine (NAC) was added to quench unreacted vcMMAE and allowed to react for 20 minutes before the resin was washed sequentially with PBS/EDTA/5% v/v DMA and 0.1M glycine pH 5.0.
[0355] The conjugates were eluted with 0.1M glycine pH 3.0 and collected into 2% v/v of 1M tris(hydroxymethyl)aminoethane (TRIS) to neutralise them.
[0356] The process was repeated in an independent second experiment using a second column/operator.
[0357] The eluted conjugates were then analysed by Hydrophobic Interaction Chromatography (
[0358] The results are summarized in Table 3 below:
TABLE-US-00004 TABLE 3 Preparation Method DAR % Aggregate Herceptin 0 0.2 Solution Phase 2.4 0.6 Column A 2.4 0.3 Column B 2.4 0.3
[0359] The data shows that when adapted to a chromatographic flow mode the conjugation of vcMMAE to Herceptin is consistent with respect to average drug loading, reduction pattern and aggregate generation. The DAR achieved in batch mode and chromatographic mode is the same when TCEP to antibody ratio is matched.
Example 4
Solid Phase Herceptin Conjugation with DM1 in Batch Mode via SMCC activation of Lysine side chains.
[0360] This example shows that immobilized antibodies can be conjugated on the side chain of lysine by modification with SMCC followed by conjugation with DM1 and that product quality is enhanced relative to the same conjugates made in solution.
[0361] Herceptin (0.5 ml of 4 mg/ml PBS, pH 7.4) was bound to 100 μl (settled resin volume) of Fabsorbent™ F1P HF resin equilibrated in PBS by mixing the resin slurry and antibody solution gently for 30 minutes. Unbound Herceptin was removed by washing the resin with PBS followed by ‘Modification Buffer’ (50 mM NaPi, 150 mM NaCl, 2 mM EDTA pH 6.7) and the resin finally re-suspended in modification buffer containing 5% v/v DMA.
[0362] The bound Herceptin was modified by adding succinimidyl-4-(N-maleimidomethyl)cyclohexyl-1-carboxylate (SMCC) to a ratio of 5 to 20 moles of SMCC per mole of Herceptin and then incubating the suspension at ambient temperature for 2 hours. Unreacted SMCC was removed by washing the resin with PBS/5% v/v DMA followed by ‘Conjugation Buffer’ (35 mM sodium citrate, 150 mM NaCl, 2 mM EDTA pH 5.0) and the resin finally re-suspended in conjugation buffer containing 3% v/v DMA.
[0363] DM1 was added to achieve 15 moles of DM1 per mole of Herceptin and the conjugation allowed to proceed for 18 hours at ambient. The resin was then washed sequentially with PBS/EDTA/5% v/v DMA and 0.1M glycine pH 5.0.
[0364] The conjugates were eluted with 0.1M glycine pH 3.0 and collected into 2% v/v of 1M tris(hydroxymethyl)aminoethane (TRIS) to neutralise them.
[0365] A solution phase conjugate of Herceptin-DM1 with an average DAR of approximately 3.5 was produced by reacting Herceptin with 7.6 moles of SMCC followed by 5 moles of DM1 per mole of Herceptin and analysed to provide a comparison of solid phase and solution phase conjugate quality. The concentration of Herceptin during the modification and conjugation reactions was 10 and 5 mg/ml respectively.
[0366] The eluted conjugates were then analysed by Size Exclusion Chromatography and UV to determine the percentage aggregate and average drug loading.
[0367] The results are summarized in Table 4 below:
TABLE-US-00005 TABLE 4 Production [Herceptin] during Method conjugation mg/ml DAR % Aggregate Solution 5 3.6 3.2 Solid Phase 20 1.7 1.8 2.6 2.8 3.5 3.0 4.8 3.5
[0368] The data shows that on solid supports lysine side-chain conjugation is possible and that the relationship between SMCC to antibody ratio and final drug loading is linear.
[0369] In addition when compared with an equivalent conjugate made in solution the solid phase conjugates show an equivalent percentage aggregation despite a four-fold increase in protein concentration during the conjugation reaction.
Example 5
Solid Phase Partial TCEP Reduction in Batch Mode Comparing Protein A Mimetic Resin, Protein L Mimetic Resin & Traditional Solution Phase
[0370] This example shows that antibody conjugation can be achieved following immobilization of antibodies on either a Protein A mimetic resin or a Protein L mimetic resin. In parallel a traditional solution phase methodology was employed using identical conditions for comparison purposes.
[0371] Herceptin (0.5 ml of 2 mg/ml PBS, pH 7.4) was bound to 100 μl (settled resin volume) of both Fabsorbentυ A1P HF and Mabsorbentυ A1P HF resins equilibrated in PBS by mixing the resin slurry and antibody solution gently for 30 minutes. Unbound Herceptin was removed by washing the resin with PBS, 2 mM EDTA and the resin finally re-suspended in 0.5 ml PBS/EDTA. The bound Herceptin was reduced by adding tris-(2-carboxyethyl)phosphine hydrochloride to a ratio of 1 to 4 moles of TCEP per mole of Herceptin and then incubating the suspension at ambient temperature for 2 hours.
[0372] vcMMAE and dimethylacetamide (DMA) were added to achieve 2.5 to 10 moles of vcMMAE per mole of Herceptin and 5% v/v DMA. The conjugations were allowed to proceed for 15 to 30 minutes at ambient temperature. N-Acetyl cysteine (NAC) was added to quench unreacted vcMMAE. After incubation for 20 minutes at ambient temperature each resin was washed sequentially with PBS/EDTA/5% v/v DMA and 0.1M glycine pH 5.0. ADC conjugates were eluted with 0.1M glycine pH 3.0 and collected into 2% v/v of 1M tris(hydroxymethyl)aminoethane (TRIS) to neutralise them.
[0373] For solution phase reactions the same reduction and conjugation conditions were employed and the final conjugates desalted into PBS via G25 batch desalting columns prior to comparative analysis.
[0374] All conjugates were all analysed by Hydrophobic Interaction Chromatography (HIC) and Size Exclusion Chromatography (SEC) to determine the percentage aggregate and average drug loading (DAR).
[0375] The data's obtained are summarized in Table 5 below:
TABLE-US-00006 TABLE 5 Fabsorbent ™ Mabsorbent ™ A1P HF A1P HF Solution Phase Average Average Average % DAR % Monomer DAR % Monomer DAR Monomer 0.9 100 0.7 100 1.3 99.6 2.2 100 1.4 100 2.4 99.3 2.8 99.8 1.7 99.6 3.4 98.9 3.5 99.7 2 99.8 4.4 98.5
[0376] The elution profiles for Herceptin-vcMMAE conjugates synthesised by solid phase means using Mabsorbent™ A1P HF resin are evidenced in
[0377] The data demonstrates that high quality antibody drug conjugates can be manufactured on either Protein A mimetic resins or Protein L mimetic resins. In addition, these data are in good agreement with earlier data (see Example 2). Both data sets demonstrate that conjugates synthesised by solid phase methods have a higher monomer content than conjugates synthesised by analogous solution phase methods when conjugates of the same average DAR are compared.
Example 6
Scalability of Solid Phase Conjugation of Antibody Drug Conjugates on Column
[0378] Flow mode syntheses are attractive for large scale manufacturing. This example evidences that solid phase synthesis of conjugates is scalable and consistent in respect of product quality and yield across various column sizes.
[0379] A series of experiments were undertaken to develop a conjugation process which achieved a DAR of 3.6±0.2 using a trastuzumab/TCEP/vcMMAE conjugation model adapted to Fabsorbent™ F1P HF resin in column/flow mode. The model was then scaled up by increasing column diameter, column length and protein loading (mg/ml) whilst maintaining factors such as linear flow rates, TCEP/vcMMAE to antibody ratio and reaction times. At small scale the released conjugates were formulated by G25 buffer exchange (PD10 or HiPrep XK16/10) and at larger scale by TFF diafiltration into 5 mM Histidine, 50 mM Trehalose, 0.01% Tween 20, at pH 6.—see Table 6 for a summary of the various conditions tested.
[0380] Fabsorbent™ F1P HF resin was prepared for column packing by washing with the column running buffer 10 mM Tris/2 mM EDTA at pH 7.5. The column was packed as a 50% slurry at 10 cm/min. A 10% overage relative to final required bed volume was used to allow for resin compression during packing.
[0381] All loading, washing, reaction and elution steps were performed at a fixed liner flow rate of 2 cm/min.
[0382] Trastuzumab antibody was supplied at a concentration of 24.1 mg/ml. Trastuzumab was diluted to 2 mg/ml and loaded onto the Fabsorbent™ F1P HF resin in 10 mM Tris/2 mM EDTA at pH 7.5 buffer to achieve the required resin loadings. After antibody loading the column was washed with 5 column volumes (CV) of 10 mM Tris/2 mM EDTA at pH 7.5 buffer. UV analysis of the load breakthrough and subsequent washes confirmed complete binding of Trastuzumab at all target loadings.
[0383] A reactant reservoir/recirculation loop external to the main column was established using a micro peristaltic pump and three way valves on the top and bottom of the main column. The reservoir volume was adjusted to achieve a 50% volume relative to the main column and this is where all process reactant were charged to.
[0384] Disulphide reduction was achieved with TCEP (2.24 equiv. wrt trastuzumab) added to the reservoir and recirculation for 2 hours at ambient temperature. The reduced trastuzumab was washed with 5 CV of 10 mM Tris/2 mM EDTA/5% DMA at pH 7.5 buffer. Conjugation was initiated by adding 10 mM vcMMAE in DMA (5 equiv.) to the reservoir and recirculating this for a total of 60 minutes at ambient temperature.
[0385] Unreacted vcMMAE was quenched by the addition of N-acetyl cysteine (NAC, 10 equiv.) to the reservoir. The resultant mixture was recirculated for 20 minutes before the final wash steps were performed.
[0386] The columns were washed with 5 CV of 10 mM Tris/2 mM EDTA/5% DMA at pH 7.5 buffer followed by 5 CV of 10 mM Tris/2 mM EDTA at pH 7.5 and then eluted using a 10 CV step elution with 0.1 M glycine, pH 3. UV spectroscopic analysis was used to determine the protein containing fractions which were then combined for final buffer exchange via G25 or TFF; depending on scale. All ADCs were formulated into 5 mM Histidine, 50 mM Trehalose, 0.01% Tween 20, at pH 6 and diluted to 1 mg/ml.
[0387] HIC, SEC and RP-HPLC chromatographic methods were used to determine the average DAR, pattern of reduction and monomer content following final formulation. Residual solvent and residual vcMMAE quantification by RP-HPLC was performed on the pooled released fractions prior to either G25 or TFF.
[0388] As a direct comparison, three solution phase conjugations were performed in a similar manner using trastuzumab and vcMMAE. Trastuzumab was pH adjusted to pH 8.2 using 500 mM borate, 25 mM EDTA. Partial reduction of disulphides was achieved by incubation of the trastuzumab with TCEP (1.94 equiv. with respect to antibody) for 90 mins at 20° C. Conjugation of reduced trastuzumab with vcMMAE (4.85 equiv.) was accomplished over 30 mins at 20° C. Excess vcMMAE was then quenched with NAC (4.85 equiv.) over 20 mins at ambient temperature. Conjugates were then purified/formulated using the same G25 column/process used for the smaller scale solid phase conjugations to afford resultant trastuzumab-vcMMAE conjugates of targeted DAR of 3.6±0.2.
[0389] The data in Table 6 demonstrates that both solution phase and solid phase syntheses can achieve a target DAR consistently. However, when compared directly to data produced from solid phase syntheses at various scales the quality of the conjugate product from solution phase is lower. The solution phase methods deliver conjugates with measureable levels of both free toxin linker and solvent. Residual toxin linker would need to be removed by further purification before the conjugate could be used in any in-vitro cell assay.
[0390] The data in Table 6 also highlights an important differentiation between solution phase and solid phase conjugation. Solid phase methodologies consistently deliver conjugates with undetectable levels of free toxin and free solvent. In essence the solid phase technique purifies away superfluous reagents through the washing of the conjugate whilst immobilised on the solid phase resin to deliver excellent purity conjugates.
[0391] Table 6 below compares and contrasts the synthesis of trastuzumab-vcMMAE conjugates synthesised by solid phase and solution phase methods.
TABLE-US-00007 TABLE 6 Column Column Sample ID Phase Scale Loading Diameter Length Formulation DAR Monomer [DMA] *1 [Toxin] *2 045_011 Solution 24 mgs NA NA 3 G25 3.5 99.9 0.0002 4.9 045_016 Solution 24 mgs NA NA 3 3.6 98.9 0.0006 4.5 045_017 Solution 24 mgs NA NA 3 3.7 98.6 0.0002 4.9 045_005 Solid 24 mgs 10 g/L 1 3 G25 3.8 99.9 0 0 045_006 Solid 24 mgs 10 g/L 1 3 3.6 99.9 0 0 045_007 Solid 24 mgs 10 g/L 1 3 3.6 99.9 0 0 045_008 Solid 53 mgs 10 g/L 1.5 3 3.6 99.9 0 0 045_009 Solid 53 mgs 10 g/L 1.5 3 3.5 99.9 0 0 045_010 Solid 150 mgs 10 g/L 2.5 3 TFF 3.7 99.9 0 0 053_007 Solid 40 mgs 10 g/L 1 5 G25 3.6 99.9 0 0 053_008 Solid 40 mgs 10 g/L 1 5 3.7 99.9 0 0 053_021 Solid 48 mgs 20 g/L 1 3 3.6 99.9 0 0 053_023 Solid 235 mgs 20 g/L 1 15 TFF 3.7 99.9 0 0 *1 [DMA] is % volume/volume, LOD = <0.0001% v/v *2 [Toxin] = free expressed as percentage of free and bound, LOD = <0.1%
MS Analysis Following Ides/EndoH/DTT Treatment
[0392] Selected conjugate samples from Table 6 were analysed my MS following treatment with Ides (FabRICATOR™, Genovis), Remove iT™ Endo S (New England Biolabs) and DTT. The overlapping ion series were then de-convoluted to give the fragment mass pattern consisting of naked and conjugated (toxin) antibody fragments from which DAR by MS could be calculated using signal intensity. The collected data is shown in Table 7 below.
TABLE-US-00008 TABLE 7 LC + Fd + Fd + Fd + DAR by DAR by Sample LC vcE Fd vcE 2vE 3vcE MS HIC 045_005 0.56 0.44 0.20 0.38 0.30 0.12 3.6 3.8 045_006 0.56 0.44 0.22 0.38 0.31 0.10 3.5 3.6 045_007 0.55 0.45 0.19 0.36 0.33 0.12 3.6 3.6 045_008 0.53 0.47 0.23 0.38 0.29 0.10 3.5 3.6 045_009 0.53 0.47 0.22 0.38 0.29 0.11 3.6 3.5 045_010 0.54 0.46 0.19 0.36 0.33 0.12 3.7 3.7 045_011 0.45 0.55 0.21 0.45 0.23 0.11 3.6 3.5 045_016 0.45 0.55 0.21 0.45 0.23 0.11 3.6 3.6 045_017 0.54 0.56 0.19 0.45 0.24 0.12 3.8 3.7
[0393] The average DARs calculated using this MS technique are consistent across the various solid phase scales employed to synthesised the trastuzumab-vcMMAE conjugates. Furthermore, DAR by MS and DAR by HIC were in excellent agreement. The pattern of reduction is consistent as shown by the consistent response fraction for naked versus conjugated LC and Fd fragments. This analysis highlights a subtle difference in the pattern of reduction between solution and solid phase conjugations. In solution there is more reduction at the HL disulphide as shown by the different average LC/LC+vcE ratios for solid phase and solution.
Cell Killing Analysis of Conjugates
[0394] Selected conjugate samples from Table 6 were analysed for potency in an antigen positive cell killing assay. SK-BR3 cell are harvested with trypsin/EDTA and then washed in assay medium and then diluted to 0.9×105/ml with more assay medium. 100 μL of this cell stock is added to each well of a 96 well plate and the plates are incubated at 37° C./5% CO.sub.2 for 3 hours to settle the cells. Samples and standards are diluted as appropriate in assay medium and added 100 μL to wells as appropriate. The cells/samples are incubated for 72 hours and then % cell cytotoxicity is measured using a commercial LDH assay kit.
[0395] In an SK-BR3 Her2 positive cell killing assay (LDH assay) the conjugates were equipotent whether made by solid phase resin or in solution. The data are summarised in Table 8 below.
TABLE-US-00009 TABLE 8 Global Global Sample Assay 1 Assay 2 Average Average SD C.V. 045_005 0.00871 0.00917 0.0089 0.001001 0.000551 5.5% 045_006 0.00934 0.0105 0.0099 045_007 0.00992 0.01036 0.0101 045_008 0.0104 0.01123 0.0108 045_009 NT 0.0102 0.0102 045_010 0.0096 0.0094 0.0095 045_011 0.0102 0.0107 0.0105 045_016 0.00983 0.0104 0.0101 045_017 0.01004 0.0102 0.0101
[0396] Assay 1 was run with duplicate samples. Assay 2 run with triplicate samples. The average column in Table 8 is the average of all 5 data points from Assay 1 & 2.
[0397] The results indicate no differences in ADC efficacies in an antigen positive cell killing assay between ADC conjugates synthesised by solid phase or solution phase means.
Example 7
Solid Phase Herceptin Conjugation with DM1 Pre-aAtivated with SMCC
[0398] This example demonstrates that immobilized antibodies can be conjugated on the side chain of lysine by modification with a pre-activated DM1-SMCC cytotoxin drug linker. This methodology is referred to as a ‘1 step approach’ to producing conjugates. Activated DM1-SMCC is prepared by incubating an excess of DM1-SH with SMCC to drive the coupling reaction to completion and then using this crude mixture for conjugation.
[0399] The thiol functionalised cytotoxin DM1 was pre-activated with the heterobifunctional crosslinker SMCC (1.6 equiv DM1 with respect to SMCC) in DMA over 5 hours at ambient temperature. A theoretical DM1-SMCC concentration was determined based on a 100% conversion of DM1 to DM1-SMCC.
[0400] Fabsorbent™ F1P HF resin (100 μl) was loaded with either 1 or 2 mgs of Herceptin and suspended in modification buffer (360 μl) composed of 50 mM NaPi, 150 mM NaCl, 2 mM EDTA at pH 6.7. DM1-SMCC drug linker was added to the slurry along with DMA to a final concentration of 10% v/v. Three different DM1-SMCC excesses were used: 5, 10 and 15 equiv. with respect to mAb bound. Conjugation reactions were agitated on a rotator for 2 hours at ambient temperature.
[0401] Post-conjugation the supernatant was removed and the resin washed sequentially with 10% v/v DMA in PBS, then PBS buffer pH 7.4 alone. Conjugates were cleaved form the resin with 0.1M succinic acid, pH 2.6 (2×500 μl with 5 min incubation at ambient temperature). Resultant conjugates were analysed by UV and SEC analysis to determine the average DAR and monomer content. The results are summarised in Table 9 below.
TABLE-US-00010 TABLE 9 mg Her DM1-SMCC % Monomer Sample bound excess DAR by UV By SEC a 1 5 1.05 99.8 b 1 10 1.94 99.7 c 1 15 2.17 99.8 d 2 5 1.25 99.7 e 2 10 2.52 99.6 f 2 15 2.96 99.6
[0402] A linear relationship was observed between DAR and DM1-SMCC excess used, with increasing DM1-SMCC leading to an increase in DAR. The DAR's can also be increased by increasing the Herceptin loading on to the solid phase resin.
[0403] Monomer levels are high with all conjugates above 99.5% irrespective of DAR.
Example 8
Application of a Chemically Modified Antibody for the Solid Phase Synthesis of an Antibody Drug Conjugate
[0404] This example demonstrates the synthesis of an ADC using a chemically modified antibody in conjunction with the solid phase conjugation technique. The antibody is firstly chemically reduced in solution prior to being incubated and bound to a solid phase resin where after the conjugation process occurs on the resin.
[0405] Herceptin antibody (1 ml of 1 mg/ml in PBS, pH 7.4, 2 mM EDTA) was reduced with the reductant 1 mM TCEP (2 equiv. wrt antibody) over a 90 min duration at ambient temperature.
[0406] Fabsorbent™ F1P HF resin (100 μl) was washed with 4× aliquots of 50 mM NaPi, pH 8 buffer. Excess buffer was removed affording damp resin to which was charged 1 ml of reduced Herceptin in PBS, pH 7.4, 2 mM EDTA. Resultant antibody resin slurry was agitated on a rotator for 30 mins at ambient temperature. The slurry was then centrifuged, the supernatant removed which was then analysed by UV to determine antibody binding by subtractive absorbance. The antibody-resin was then washed with 1 ml PBS, pH 7.4, 2 mM EDTA buffer. The wash fraction was also analysed to confirm the overall concentration of antibody bound to the resin.
[0407] Reduced antibody resin was suspended in PBS pH 7.4, 2 mM EDTA buffer (950 μl) and DMA (4.6 μl). Cytotoxin drug linker mcF or vcMMAE in DMA (10 mM, 3.3 μl) was charged to the resin slurry to afford an overall 5% v/v DMA in PBS pH 7.4, 2 mM EDTA media. The conjugation reaction proceeded for 30 mins at ambient temperature with gentle agitation on a rotator.
[0408] The conjugation reaction was quenched by the addition of NAC (10 mM, 3.3 μl) to the slurry and gently agitating the resultant mixture for 20 mins at ambient temperature.
[0409] The resin was then filtered and washed with 2×5% v/v DMA in PBS, pH 7.4.
[0410] ADC conjugates were released from solid phase resins by treatment with 0.1M glycine, pH 3 (980 μl) for 5 mins at ambient temperature. Resin slurries were then centrifuged and the supernatant removed. A single charge of 20 ml of 1M Tris buffer was added to the supernatant to make a 1 ml sample suitable for UV analysis to determine recovery yield.
[0411] Samples were then analysed directly; prior to desalt on G25 packed columns, by Hydrophobic Interaction Chromatography (HIC) to calculate Drug to Antibody Ratio (DAR).
[0412]
Example 9
Solid Phase Site-Specific Conjugation Using a Recombinantly Engineered Thiol-Antibody
[0413] This example exemplifies the synthesis of antibody drug conjugates using solid phase resins with recombinantly engineered thiol-antibodies. In this example the recombinantly engineered antibody contains 2 additional cysteine residues that facilitates a site-specific conjugation technique similar to that of ThioMab antibody technology (Genentech).
[0414] A 1 ml solution of Herceptin with engineered cysteines (V205C Kabat numbering) was supplied in formulation buffer comprised of 5 mM histidine, 50 mM trehalose and 0.01% v/v PS20 (concentration 20 mg/ml). The 1 ml antibody solution was diluted with 4 ml of 10 mM Tris, pH 7.5 buffer. The resultant antibody solution was incubated with Fabsorbent™ F1P HF resin (1 ml, settled resin volume) pre-equilibrated in 10 mM Tris, pH 7.5 buffer. The antibody was bound to the resin with gentle rotation of the slurry on a rotator for 10 minutes at ambient temperature.
[0415] The bound Herceptin with engineered cysteines was completely reduced by treatment with a large excess of the reductant DTT (20 equiv. wrt antibody). The reducing suspension was gently agitated on a rotator for 16 hours at ambient temperature. The resin was then washed with 2×5 ml 50 mM Tris 2.5 mM EDTA, pH 8 buffer followed by 2×5 ml 10 mM Tris, pH 7.5 buffer to remove all traces of DTT. The resin was finally re-suspended in 5 ml of 10 mM Tris, pH 7.5 buffer.
[0416] The immobilised antibody with engineered cysteines was then re-oxidised by the addition of dehydroascorbic acid (dhAA) in DMA (10 equiv. wrt antibody) and the resin slurry gently agitated on a rotator over a 1 hr period at ambient temperature. A charge of the cytotoxin drug linker vcMMAE in DMA (2.5 equiv. wrt antibody) was then added to the immobilised antibody with engineered cysteines on resin to achieve a final composition of 5% v/v DMA in buffer mixture. Conjugation proceeded for 1 hr at ambient temperature with rotation.
[0417] Excess vcMMAE was then removed from the resin slurry by washing the resin with 3×5 ml 5% v/v DMA in 10 mM Tris, pH 7.5 buffer then 3×5 ml 10 mM Tris, pH 7.5 buffer.
[0418] ADC conjugate was released from the resin by incubation with 0.1M glycine, pH 2.96 (5 ml) with rotation for 10 min. The released Herceptin with engineered cysteines-vcMMAE conjugate was then immediately formulated via a High Trap Desalting Column (GE Healthcare) into 5 mM histidine, 50 mM Trehalose, 0.01% v/v PS20, pH 6 buffer.
[0419] Released conjugate samples were then analysed by Size Exclusion Chromatography (SEC) to determine monomer level. Hydrophobic Interaction Chromatography (HIC) and RP-PLRP were used to calculate Drug to Antibody Ratio (DAR). The data for these analyses is reported in
[0420] Analytical SEC chromatography demonstrates that the solid phase technique in conjunction with a recombinantly modified antibody affords ADC conjugates in excellent purity as evidenced by very high monomer content. The HIC profile also evidences that an average DAR of 2.1 and distribution of DAR 0, 1, 2 and higher are consistent with published results for Thiomabs. This demonstrates that the solid phase can be used to generate site-specific ADCs incorporating engineered cysteine residues within an engineered antibody.
Example 10
Solid Phase Herzuma® and Cetuximab (Erbitux®) Conjugation with DM1-SMCC
[0421] This example demonstrates the synthesis of an antibody drug conjugate in a ‘1 step approach’. Herzuma® is a biosimilar monoclonal antibody of trastuzumab (Herceptin, Roche). Herein we demonstrate that the antibody drug conjugate Herzuma®-DM1 can be synthesised using a solid phase conjugation approach via the use of the pre-qualified activated DM1 toxin-linker MCC-DM1.
[0422] Three concentrations of Herzuma® at 2 mg/ml, 4 mg/ml and 6 mg/ml were prepared from a stock sample (25.6 mg/ml) by dilution with PBS, pH 7.4 buffer. In parallel, three concentrations of Cetuximab (Erbitux®) at 2 mg/ml, 4 mg/ml and 6 mg/ml were also prepared from a stock sample (15.5 mg/ml) by dilution with PBS, pH 7.4 buffer. UV absorbance measurements of each sample were taken at 280 & 320 nm.
[0423] Six separate samples of Fabsorbent™ F1P HF resin (100 μl) were washed with 4×50 μl aliquots of 50 mM NaPi, pH 8 buffer and the supernatants removed. 0.5 ml aliquots of each of the antibody concentrations were incubated with resin at ambient temperature with gentle agitation on a rotator over a 1 hr period. Separately, each resin sample was centrifuged and the supernatant removed. Each supernatant was analysed by UV at 280 & 320 nm. The loading of antibody onto each resin was determined by subtractive UV. Each resin was then washed with 1× aliquot of PBS to remove any superfluous antibody.
[0424] Conjugation buffer was prepared at three separate pH's: 0.1M NaPi, pH 7.5; 0.1M NaPi, pH 8 & 0.1M NaHCO.sub.3, pH 8.5. Resin samples with bound antibody were incubated separately with each buffer.
[0425] To each slurry was charged the toxin-linker MCC-DM1 in DMA. A range of excess equivalents of MCC-DM1 were used from 5 to 10 equivalents wrt antibody. Overall, the MCC-DM1 charge afforded conjugation media's of 5% v/v DMA in buffers.
[0426] Conjugation reactions were performed over 2 hrs at ambient temperature with gentle agitation on a rotator.
[0427] After this durations the supernatants were removed and each resin washed with 4×50 μl aliquots of 5% v/v DMA in PBS, pH 7.4 followed by 3×50 μl aliquots of PBS, pH 7.4.
[0428] Antibody drug conjugates were removed from the solid phase resins by incubation of each resin in 50 μl of 50% v/v propylene glycol in 0.1M glycine, pH 3 over 30 mins at ambient temperature. Supernatants were collected separately and analysed by SEC at 214 nm to determine yield and monomer content. SEC analysis at 252 nm & 280 nm facilitated the calculation of DAR. The data for conjugates immediately following removal from solid phase resin is shown in Table 10 (ELUTED).
TABLE-US-00011 TABLE 10 % % mg's MCC- monomer monomer mAB DM1 DAR (214 nm) DAR (214 nm) Sample mAb bound pH excess ELUTED FORMULATED A Herzuma ® 0.99 7.5 5 0.6 99.6 0.6 99.9 B Herzuma ® 1.98 8 7.5 1.6 99.5 1.6 99.8 C Herzuma ® 2.91 8.5 10 3.7 98.5 3.8 99.4 D Cetuximab 1.00 7.5 5 0.5 99.5 0.5 99.7 E Cetuximab 1.98 8 7.5 1.4 99.1 1.4 99.4 F Cetuximab 2.93 8.5 10 3.9 98.2 3.8 98.5
[0429] All conjugates were then desalted into formulation buffer. For Herzuma® conjugates a formulation buffer of 5 mM histidine, 50 mM trehalose, 0.01% PS20, pH 6 was employed. For Cetuximab a formulation buffer of 20 mM Succinate, 150 mM NaCl, 2 mM EDTA, pH 5.5 was employed.
[0430] All conjugates were desalted using NAP5 columns. Post-desalt samples were then re-analysed by SEC chromatography to determine monomer purity on the final materials. Data for the final conjugates in formulation buffer is shown in Table 10 (FORMULATED).
[0431] SEC traces for Herzuma®-MCC-DM1 and Cetuximab-MCC-DM1 conjugates are evidenced in
[0432] The data clearly shows that consistently high monomeric products can be achieved using the solid phase conjugation technique. By varying the toxin-linker excess a range of conjugates of varying DAR can be easily achieved.