Protecting Group Comprising a Purification Tag

Abstract

The present invention relates to compounds comprising a protecting group moiety-tag moiety conjugate, a method of purification and monoconjugates obtained from such method of purification.

Claims

1. A compound having a moiety of formula (I): ##STR00061## wherein: the dashed line indicates attachment to the rest of the compound; T is a tag moiety; and PG is a protecting group moiety.

2. The compound of claim 1; wherein PG is utilized for the reversible protection of an amine functional group and wherein PG comprises a moiety of formula (Aa), (Ab), or (Ac): ##STR00062## wherein: the dashed line marked with the asterisk indicates attachment to T and the unmarked dashed line indicates attachment to the nitrogen of an amine functional group; n is 0, 1, 2, 3, or 4; X is a chemical bond or a spacer; Y.sub.1 is selected from the group consisting of O and S; Y.sub.2 is selected from the group consisting of O and S; Y.sub.3 and Y.sub.5 are independently of each other selected from the group consisting of O and S; Y.sub.4 is selected from the group consisting of O, NR.sup.5, and C(R.sup.6R.sup.6a); R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.6a are independently of each other selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; W is selected from the group consisting of C.sub.1-20, alkyl optionally interrupted by one or more groups selected from the group consisting of O, S and N(R.sup.7); -Nu is a nucleophile selected from the group consisting of N(R.sup.7R.sup.7a), N(R.sup.7OH), N(R.sup.7)N(R.sup.7aR.sup.7b), S(R.sup.7), COOH, ##STR00063## -Nu is a nucleophile selected from the group consisting of N(R.sup.7), N(OH), N(R.sup.7)N(R.sup.7a), S, C(O)O, ##STR00064## wherein: the unmarked dashed line indicates attachment to the rest of the moiety of formula (Ac); and the dashed line marked with the asterisk indicates attachment to T; Ar is selected from the group consisting of: ##STR00065## wherein: dashed lines indicate attachment to the rest of the prodrug, Z.sup.1 is selected from the group consisting of O, S, and N(R.sup.7); and Z.sup.2 is N(R.sup.7); and R.sup.7, R.sup.7a, and R.sup.7b are independently of each other selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the moiety of formula (Aa), (Ab), or (Ac) is optionally further substituted.

3. The compound of claim 1; wherein PG is utilized for the reversible protection of an amine functional group; and wherein PG of formula (I) preferably comprises a moiety of formula (C): ##STR00066## wherein: the dashed line marked with the asterisk indicates attachment to T of formula (I) and the unmarked dashed line indicates attachment to the nitrogen of an amine functional group; X is C(R.sup.4R.sup.4a), N(R.sup.4), O, C(R.sup.4R.sup.4a)C(R.sup.5R.sup.5a), C(R.sup.5R.sup.5a)C(R.sup.4R.sup.4a), C(R.sup.4R.sup.4a)N(R.sup.6), N(R.sup.6)C(R.sup.4R.sup.4a), C(R.sup.4R.sup.4a)O, or OC(R.sup.4R.sup.4a); X.sup.1 is C or S(O); X.sup.2 is C(R.sup.7, R.sup.7a) or C(R.sup.7, R.sup.7a)C(R.sup.8, R.sup.8a); R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8, and R.sup.8a are independently selected from the group consisting of H and C.sub.1-6 alkyl; optionally, one or more of the pair(s) R.sup.1a/R.sup.4, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.4a/R.sup.5a, and R.sup.7a/R.sup.8a form a chemical bond; optionally, one or more of the pair(s) R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a, R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, and R.sup.8/R.sup.8a are joined together with the atom to which they are attached to form a C.sub.3-8 cycloalkyl or a 4-membered to 7-membered heterocyclyl; optionally, one or more of the pair(s) R.sub.1/R.sup.4, R.sup.1/R.sup.5, R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.7/R.sup.8, and R.sup.2/R.sup.3 are joined together with the atoms to which they are attached to form a ring A; optionally, R.sup.3/R.sup.3a are joined together with the nitrogen atom to which they are attached to form a 4-membered to 7-membered heterocycle; A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8-membered to 11-membered heterobicyclyl; provided that one hydrogen of R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8, or R.sup.8a is replaced by -T of formula (I); and wherein the moiety of formula (C) is optionally further substituted.

4. The compound of claim 1; wherein PG is utilized for the reversible protection of a thiol functional group; and PG is selected from the group consisting of: ##STR00067## wherein: the dashed line marked with an asterisk indicates attachment to T and the unmarked dashed line indicates attachment to the rest of the compound; Ar is an aromatic moiety which is optionally further substituted; R.sup.01, R.sup.03, and R.sup.04 are independently of each other a chemical bond or is C.sub.1-50 alkyl, C.sub.2-50 alkenyl, or C.sub.2-50 alkynyl, wherein: C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more R.sup.3, which are the same or different; and C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, C(O)O, O, C(O), C(O)N(R.sup.4), S(O).sub.2N(R.sup.4), S(O)N(R.sup.4), S(O).sub.2, S(O), N(R.sup.4)S(O).sub.2N(R.sup.4) , S, N(R.sup.4), OC(O)R.sup.4, N(R.sup.4)C(O), N(R.sup.4)S(O).sub.2, N(R.sup.4)S(O), N(R.sup.4)C(O)O, N(R.sup.4)C(O)N(R.sup.4), and OC(O)N(R.sup.4R.sup.4a); R.sup.02 is H, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, or C.sub.2-50 alkynyl, wherein: C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more R.sup.3, which are the same or different; and C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, C(O)O, O, C(O), C(O)N(R.sup.4), S(O).sub.2N(R.sup.4), S(O)N(R.sup.4), S(O), S(O), N(R.sup.4)S(O).sub.2N.sup.4a), S, N(R.sup.4), OC(O)R.sup.4, N(R.sup.4)C(O), N(R.sup.4)S(O).sub.2, N(R.sup.4)S(O), N(R.sup.4)C(O)O, N(R.sup.4)C(O)N(R.sup.4), and OC(O)N(R.sup.4R.sup.4a); Q is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8 membered to 11-membered heterobicyclyl, wherein T is optionally substituted with one or more R.sup.3, which are the same or different; R.sup.3 is halogen, CN, oxo (O), COOR.sup.5, OR.sup.5, C(O)R.sup.5, C(O)N(R.sup.5R.sup.5a), S(O).sub.2N(R.sup.5R.sup.5a), S(O)N(R.sup.5R.sup.5a), S(O).sub.2R.sup.5, S(O)R.sup.5, N(R.sup.5) S(O).sub.2N(R.sup.5aR.sup.5b), SR.sup.5, N(R.sup.5R.sup.5a), NO.sub.2, OC(O)R.sup.5, N(R.sup.5)C(O)R.sup.5a, N(R.sup.5)S(O).sub.2R.sup.5a, N(R.sup.5)S(O)R.sup.5a, N(R.sup.5)C(O)OR.sup.5a, N(R.sup.5)C(O)N(R.sup.5aR.sup.5b), OC(O)N(R.sup.5R.sup.5a), or C.sub.1-6 alkyl, wherein: C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, and R.sup.5b are independently selected from the group consisting of H or C.sub.1-6 alkyl, wherein: C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

5. The compound of claim 4; wherein Ar is selected from the group consisting of: ##STR00068## ##STR00069## wherein: dashed lines marked with an asterisk indicate attachment to T of formula (I) and the unmarked dashed lines indicate attachment to the rest of PG; W is independently of each other O, S, or N; and W is N; and wherein Ar is optionally substituted with one or more substituent(s) independently selected from the group consisting of NO.sub.2, Cl, and F.

6. The compound of claim 4; wherein PG is: ##STR00070## wherein: the dashed line marked with an asterisk indicates attachment to T; and the unmarked dashed line indicates attachment to the rest of the compound.

7. The compound of claim 1; wherein T comprises at least one positive or negative charge.

8. The compound of claim 1; wherein T comprises at least one positive charge under aqueous conditions at pH 3 to 9.

9. The compound of claim 8; wherein the at least one positive charge is provided by an ammonium, phosphonium, or tertiary amine.

10. The compound of claim 1; wherein T comprises: a polyamide comprising at least one component selected from the group consisting of tertiary amine, quaternary ammonium residue, and protonated ammonium residue; and optionally further functional groups.

11. The compound of claim 1; wherein T comprises a moiety of formula (a): ##STR00071## wherein: the dashed line indicates attachment to the rest of the compound; R.sup.1, R.sup.1a, R.sup.1b, R.sup.2, R.sup.2a, R.sup.2b, R.sup.3, R.sup.3a, R.sup.3b, R.sup.4, R.sup.4a, and R.sup.4b are independently of each other H or methyl; each m is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; each n is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; each x is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; each y is independently of each other 0, 1, 2, 3, 4, 5, 6, 7, or 8; and SP is a spacer moiety.

12. The compound of claim 1; wherein T comprises a moiety of formula (b): ##STR00072## wherein: the dashed line indicates attachment to PG of formula (I); q is 0 or 1; and SP is a spacer moiety.

13. The compound of claim 1, wherein the compound comprises further comprising: a moiety of formula compound comprising a moiety of formula (IIa): ##STR00073## wherein: the dashed line indicates attachment to the rest of the compound; T and PG are as defined in claim 1; L.sup.2 is a chemical bond or a spacer moiety; L.sup.1 is a reversible prodrug linker moiety.

14. The compound of claim 13; wherein L.sup.2 is a spacer moiety.

15. The compound of claim 13; wherein L.sup.2 of formula (IIa) is selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y1), S(O).sub.2N(R.sup.y1), S(O)N(R.sup.y1), S(O).sub.2, S(O), N(R.sup.y1)S(O).sub.2N(R.sup.y1a), S, N(R.sup.y1), OC(OR.sup.y1)(R.sup.y1a), N(R.sup.y1)C(O)N(R.sup.y1a), OC(O)N(R.sup.y1), C.sub.1-50alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl, wherein: -T-, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally substituted with one or more R.sup.y2, which are the same or different; and C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y3), S(O).sub.2N(R.sup.y3), S(O)N(R.sup.y3), S(O).sub.2, S(O), N(R.sup.y3)S(O).sub.2N(R.sup.y3a), S, N(R.sup.y3), OC(OR.sup.y3) (R.sup.y3a), N(R.sup.y3)C(O)N(R.sup.y3a), and OC(O)N(R.sup.y3a); wherein: R.sup.y1 and R.sup.y1a are independently of each other selected from the group consisting of H, -T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl, wherein -T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally substituted with one or more R.sup.y2, which are the same or different; and C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y4), S(O).sub.2N(R.sup.y4), S(O)N(R.sup.y4), S(O).sub.2, S(O), N(R.sup.y4)S(O).sub.2N(R.sup.y4a), S, N(R.sup.y4), OC(OR.sup.y4)(R.sup.4a), N(R.sup.y4)C(O)N(R.sup.y4a), and OC(O)N(R.sup.y4); each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3-membered to 10-membered heterocyclyl, 8-membered to 11-membered heterobicyclyl, 8-membered to 30-membered carbopolycyclyl, and 8-membered to 30-membered heteropolycyclyl, wherein: each T is independently optionally substituted with one or more R.sup.y2, which are the same or different; R.sup.y2 is selected from the group consisting of halogen, CN, oxo (O), COOR.sup.y5, OR.sup.y5, C(O)R.sup.y5, C(O)N(R.sup.y5R.sup.y5a), S(O) N(R.sup.y5R.sup.y5), S(O)N(R.sup.y5R.sup.y5a), S(O).sub.2R.sup.y5, S(O)R.sup.y5, N(R.sup.y5) S(O).sub.2N(R.sup.y5aR.sup.y5b), SR.sup.y5, N(R.sup.y5R.sup.y5a), NO.sub.2, OC(O)R.sup.y5, N(R.sup.y5)C(O)OR.sup.y5a, N(R.sup.y5)S(O).sub.2R.sup.y5a, N(R.sup.y5)S(O)R.sup.y5a, N(R.sup.y5)C(O)OR.sup.y5, N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b), OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl, wherein: C.sub.1, alkyl is optionally substituted with one or more halogen, which are the same or different; and each R.sup.y3, R.sup.y3a, R.sup.y4, R.sup.y4a, R.sup.y5, R.sup.y5a, and R.sup.y5b is, independently of each other, selected from the group consisting of H, and C.sub.1, alkyl; wherein: C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

16. The compound of claim 13; wherein L.sup.1 comprises a moiety of formula (b-iii): ##STR00074## wherein: the dashed line with marked with the asterisk indicates attachment to L.sup.2 of formula (IIa); the unmarked dashed line indicates attachment to the rest of the compound comprising the moiety of formula (IIa); and the moiety of formula (b-iii) is attached to the rest of the compound through an amine functional group provided by said rest of the compound by forming an amide linkage between L.sup.1 and the rest of the compound comprising the moiety of formula (IIa); and wherein: X of formula (b-iii) is C(R.sup.4R.sup.4a), N(R.sup.4), O, C(R.sup.4R.sup.4a)C(R.sup.5R.sup.5a), C(R.sup.5R.sup.5a)C(R.sup.4R.sup.4a), C(R.sup.4R.sup.4a)N(R.sup.6), N(R.sup.6)C(R.sup.4R.sup.4a), C(R.sup.4R.sup.4a)O, or OC(R.sup.4R.sup.4a); X.sup.1 of formula (b-iii) is C or S(O); X.sup.2 of formula (b-iii) is C(R.sup.7, R.sup.7a) or C(R.sup.7, R.sup.7a)C(R.sup.8, R.sup.8a); X.sup.3 of formula (b-iii) is O, S, or NCN; R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.7, R.sup.7a, R.sup.8, and R.sup.8a of formula (b-iii) are independent of each other selected from the group consisting of H and C.sub.1-4 alkyl; optionally, one or more of the pairs R.sup.1a/R.sup.4a, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, and R.sup.7a/R.sup.8a of formula (b-iii) form a chemical bond; optionally, one or more of the pairs R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a, R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, and R.sup.8/R.sup.8a of formula (b-iii) are joined together with the atom to which they are attached to form a C.sub.3-10 cycloalkyl or a 4-membered to 7-membered heterocyclyl; optionally, one or more of the pairs R.sup.1/R.sup.4, R.sup.1/R.sup.5, R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.4/R.sup.6, R.sup.7/R.sup.8, and R.sup.2/R.sup.3 of formula (b-iii) are joined together with the atoms to which they are attached to form a ring A; optionally, R.sup.3/R.sup.3a of formula (b-iii) are joined together with the nitrogen atom to which they are attached to form a 4-membered to 7-membered heterocycle; A of formula (b-iii) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8-membered to 11-membered heterobicyclyl; and wherein the moiety of formula (b-iii) is: substituted with L.sup.2 of formula (IIa); and optionally further substituted; provided that: the hydrogen marked with the asterisk in formula (b-iii) is not replaced; and R.sup.3 and R.sup.3a of formula (b-iii) are, independently of each other, H or are connected to N through an SP.sup.3-hybridized carbon atom.

17. The compound of claim 1; wherein the compound is of formula (IIIa):
(T-PG-L.sup.2-L.sup.1).sub.x-PM(IIIa); wherein: T and PG are as defined in claim 1; L.sup.2 is a chemical bond or a spacer moiety; L.sup.1 reversible prodrug linker moiety. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and PM is a moiety having a molecular weight of at least 1 kDa.

18. The compound of claim 17; wherein PM is a protein.

19. The compound of claim 17; wherein PM comprises and antibody or antibody fragment.

20. A method of purification, comprising the steps of: (i) providing a mixture comprising a multitude of conjugates selected from the group consisting of formula (III) and formula (IIIa) which differ by their value for x, where the formulas (III) and (IIIa) are:
(T-PG-X.sup.1-L-X.sup.2).sub.x-PM(III); and
(T-PG-L.sup.2-L.sup.1).sub.x-PM(IIIa); wherein: each T is, independently of each other, a tag moiety; and each PG is, independently of each other, a protecting group moiety; X.sup.1 and X.sup.2 are each, independently of each other, a linkage; L and L.sup.2 are each, independently of each other, a chemical bond or a spacer moiety; L.sup.1 is a reversible prodrug linker moiety; each x is, independently of each other, an integer from 1-20; and each PM is, independently from each other, a moiety having a molecular weight of at least 1 kDa; (ii) subjecting the mixture of step (i) to a purification method suitable for the tag moiety T; and (iii) isolating a fraction that comprises compounds of formula (III) or (IIIa) in which at least 80% of all compounds of formula (III) or (IIIa) have the same value for x.

21. The method of claim 20; wherein T comprises a charged moiety and the purification method of step (ii) is ion exchange chromatography.

22. A monoconjugate obtained from the method of claim 20, in which x in step (iii) is 1.

Description

[0404] Another aspect of the present invention is a monoconjugate obtainable from the method of the present invention in which x in step (iii) is 1.

[0405] FIG. 1: Chromatogram (based on absorption at 280 nm) of CIEC purification of tagged Lucentis-linker monoconjugate 4b. 4b could be separated from Lucentis as well as Lucentis-linker bisconjugate. The observed two peaks at 15 cv and 18 cv were identified as 4b by mass spectrometry. Conductivity is shown as grey curve.

[0406] FIG. 2: Chromatogram (based on absorption at 280 nm) of CIEC purification of tagged Lucentis-linker monoconjugate 6b. 6b could be separated from Lucentis as well as tagged Lucentis-linker bisconjugate. The observed two peaks at 95 mL and 110 mL were identified as 6b by mass spectrometry. Conductivity is shown in grey.

MATERIALS AND METHODS

[0407] Lucentis and Ranibizumab are used synonymously throughout the following examples.

[0408] Materials:

[0409] TBTU, HATU, PyBOP, and Fmoc-L-Asp(OtBu)-OH were purchased from Merck Biosciences GmbH, Schwalbach/Ts, Germany.

[0410] Boc-Lys(Boc)-OSu was purchased from Senn chemicals AG, Dielsdorf, Switzerland. Fmoc-N-Me-L-Asp(OtBu)-OH was purchased from Bachem, Bubendorf, Switzerland. 1,9-bis-Boc-1,5,9-triazanonan was purchased from PolyPeptide Laboratories A/S, Hillerod, Denmark.

[0411] (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate was purchased from Chemzon Scientific Inc., Lachine, QC, Canada.

[0412] All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

[0413] Methods:

[0414] Reactions were performed with dry solvents (DCM, THF, ACN, DMF, dioxane, MeOH, toluene) stored over molecular sieve purchased from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany. Generally, reactions were stirred at room temperature and monitored by HPLC/MS or TLC.

[0415] RP-HPLC was done on a 10020 mm or 10040 mm C18 ReproSil-Pur 300 ODS-3 5 column (Dr. Maisch, Ammerbuch, Germany) or XBridge BEH300 C18 OBD Prep 10 m 30150 mm or 5 m 10150 mm (Waters, Eschborn, Germany) connected to a Waters 600 or 2535 HPLC System and Waters 2487 or 2489 Absorbance detector, respectively. Linear gradients of solution A (0.1% TFA in H.sub.2O) and solution B (0.1% TFA in acetonitrile) were used. HPLC fractions containing product were combined and lyophilized.

[0416] Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane, ethyl acetate, and methanol as eluents. Products were detected at 254 nm. For products showing no absorbance above 240 nm fractions were screened by LC/MS.

[0417] Analytical ultra-performance LC (UPLC) was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.150 mm, 1.7 m particle size) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific.

[0418] HPLC-Electrospray ionization mass spectrometry (HPLC-ESI-MS) was performed on a Waters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer or Waters Micromass ZQ both equipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.150 mm, 300 , 1.7 m, flow: 0.25 mL/min; solvent A: UPH.sub.20+0.04% TFA, solvent B: UP-Acetonitrile+0.05% TFA.

[0419] Buffer exchange was performed on a HiTrap or HiPrep column (GE Healthcare) connected to an Aekta Purifier system.

[0420] Cationic ion exchange chromatography was performed either on a Source 15S 4.6/100 column or on a Source 15 S 6 mL column connected to an Aekta Purifier system using 20 mM MES, pH 5.7 and 20 mM MES, 500 mM NaCl, pH 5.7 as mobile phase A and B, respectively.

EXAMPLE 1

[0421] Synthesis of Linker Reagent 1k

[0422] Linker reagent 1k was synthesized according to the following scheme:

##STR00051##

[0423] N-Boc-ethylenediamine (0.77 g, 4.8 mmol) was dissolved in DCM (15 mL) and 6-tritylmercaptohexanoic acid (2.25 g, 5.76 mmol) and PyBOP (3.0 g 5.76 mmol) were added with stirring. DIPEA (2.52 ml, 14.4 mmol) was added and the reaction stirred for 1 h. The reaction was diluted with diethyl ether (150 mL) and washed with slightly basic brine (330 mL, prepared from 100 mL brine and 3 mL 0.1 M aq. NaOH). The organic phase was washed once more with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo and purified using flash chromatography to give 1a as a white foam. Yield: 2.55 g (4.79 mmol, 99%) MS: m/z 555.24=[M+Na].sup.+, (calculated=555.27).

[0424] 1a (2.55 g, 4.79 mmol) was dissolved in THF (26 mL) and transferred into an oven-dried argon filled round-bottom flask. BoraneTHF complex in THF (1 M, 17.7 mL, 17.71 mmol) was added and the reaction stirred for 15 h. MeOH (5.4 mL) was added slowly and N,N-dimethyl ethylenediamine (3.11 mL, 28.8 mmol) was added and the reaction refluxed for 2.5 h. After cooling the reaction was diluted with ethyl acetate and washed with sat. sodium bicarbonate solution (2125 mL) and brine (1125 mL). The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo to give 1b which was used without further purification in the next step. Yield: 2.38 g (4.59 mmol, 96%) MS: m/z 519.27=[M+H].sup.+, (calculated=519.31).

[0425] 1b (1.19 g 2.29 mmol) was dissolved in DCM and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (1.02 g, 3.44 mmol) and 2,4,6-collidine (1.36 mL, 10.32 mmol) were added and the reaction stirred for 23 h. The reaction was concentrated in vacuo and purified using flash chromatography to give 1c. Yield: 1.19 g (1.77 mmol, 79%) MS: m/z 697.18=[M+Na].sup.+, (calculated=697.29).

[0426] 1c (0.5 g, 0.74 mmol) was dissolved in DCM (2.5 mL) and triphenylmethanol (0.19 g, 0.74 mmol) and TFA (2.5 mL) were added. The reaction was stirred for 40 min, concentrated in a stream of argon and dried in vacuo (<0.1 mbar). The residue was dissolved in ACN/water (7:3 v/v, 10 mL) and purified by RP-HPLC to give 1d (0.50 g, 0.84 mmol, 114%). MS: m/z 575.33=[M+H].sup.+, (calculated=575.26).

[0427] 2-Chlorotritylchloride resin (1.22 mmol/g, 0.87 g, 1 mmol) was weighted into a 10 ml syringe with frit. The resin was swollen with 5 mL DCM and washed with DCM (54 mL). N-Fmoc-L-Asp(OtBu)-OH (1.1 g, 2.7 mmol) was dissolved in DCM (5 mL) and DIPEA (0.66 mL, 3.78 mmol) was added and the solution drawn into the syringe. The syringe was agitated for 1 h. MeOH (0.5 mL) was drawn into the syringe and the syringe agitated for 15 min. The resin was washed 5 times with DCM (4 mL) and 5 times with DMF (5 mL). The resin was agitated 3 times for 5 min with DMF:DBU:piperidine (96:2:2 v/v/v 4 mL). The resin was washed 5 times with DMF (4 mL). Acetic anhydride (0.51 mL, 5.4 mmol) and DIPEA (1.9 mL, 10.8 mmol) were dissolved in DMF (6 mL) and the solution was drawn into the syringe and the syringe agitated for 15 min. The resin was washed 5 times with DMF (4 mL), 5 times with DCM (4 mL). A solution of HFIP/DCM ( v/v, 5 mL each) were drawn into the syringe and the syringe agitated 3 times for 10 min. The collected filtrates were concentrated in vacuo. Crude 1e (0.29 g, 1.23 mmol, 114%) was used without further purification in the next step. MS: m/z 254.38=[M+Na].sup.+, (calculated=254.12).

[0428] 1e (65 mg, 0.28 mmol) was dissolved in DCM (3 mL) and PyBOP (0.18 g, 0.34 mmol) and DIPEA (0.15 mL, 0.84 mmol) were added. 1d (0.18 g, 0.31 mmol) was dissolved in DCM (3 mL) and added to the reaction. The reaction was stirred for 1 h and concentrated in vacuo. The residue was purified by RP-HPLC to give 1f (97 mg, 0.12 mmol, 44%). MS: m/z 810.02=[M+Na].sup.+, (calculated=810.34).

[0429] 1f (200 mg, 0.25 mmol) were dissolved in HFIP (5 mL) and TES (0.3 mL) and AcOH (0.3 mL) were added and the reaction stirred for 75 min. The solvents were removed in a stream of argon and the residue dissolved in ACN/water (9:1 v/v) and purified by RP-HPLC to give 1g (95 mg, 0.17 mmol, 68%). MS: m/z 568.28=[M+Na].sup.+, (calculated=568.23).

[0430] 1g (95 mg, 0.17 mmol) was dissolved in ACN/water (1:1 v/v, 3 mL) and 6, 6-dithiodinicotinic acid (107 mg, 0.35 mmol) was added. ACN (1 mL) was added to improve solubility and pH 7.4 sodium phosphate buffer (0.5 M, 1.5 mL) was added to adjust the pH. The reaction was stirred for 45 min and the reaction mixture directly purified by RP-HPLC to give 1h (103 mg, 0.15 mmol, 85%). MS: m/z 699.14=[M+H].sup.+, (calculated=699.22).

[0431] Fmoc protected Sieber amide resin (0.74 mg, 0.45 mmol, 0.61 mmol/g) was weighed into a 20 mL syringe equipped with a filter frit. The resin was swollen with DMF (210 mL, 215 min) and the Fmoc protecting group was afterwards removed by shaking the resin with DMF/piperidine/DBU (98:1:1, v/v/v, 28 mL, 15 min, 110 min). After intensive washing with DMF (108 mL) the first coupling was accomplished by incubating the resin for 1 h with a solution of Fmoc-L-Lys(Me.sub.3Cl)OH (0.5 g, 1.12 mmol), HOBt (0.17 g, 1.12 mmol), HATU (0.43 g, 1.12 mmol) and DIPEA (0.78 mL, 4.5 mmol) in DMF (4 mL). After intense washing with DMF (108 mL) the terminal Fmoc protecting group was removed by treating the resin with DMF/piperidine/DBU (98:1:1, v/v/v, 28 mL, 15 min, 110 min). The resin was washed with DMF (108 mL) and the second amino acid was attached by incubating the resin for 1 h with a solution of Fmoc-L-Lys(Me.sub.3Cl)OH (0.5 g, 1.12 mmol), HOBt (0.17 g, 1.12 mmol), TBTU (0.36 g, 1.12 mmol) and DIPEA (0.78 mL, 4.5 mmol) in DMF (4 mL). After washing with DMF (108 mL) the Fmoc protecting group was removed by incubation with DMF/piperidine/DBU (98:1:1, v/v/v, 28 mL, 15 min, 110 min). After washing with DMF (108 mL) the amount of resin was halved and one part of the resin was kept aside. The next coupling was accomplished by incubating the resin for 1 h with a solution of Fmoc-L-Lys(Me.sub.3Cl)OH (0.25 g, 0.56 mmol), HOBt (86 mg, 0.56 mmol), TBTU (0.18 g, 0.56 mmol) and DIPEA (0.39 mL, 2.25 mmol) in DMF (2 ml). After washing with DMF (104 mL) and subsequent deprotection with DMF/piperidine/DBU (98:1:1, v/v/v, 24 mL, 15 min, 110 min) and washing with DMF (104 mL) the resin was treated with a coupling solution of Fmoc-Ado-OH (0.22 g, 0.56 mmol), HOBt (86 mg, 0.56 mmol), TBTU (0.18 g, 0.56 mmol) and DIPEA (0.39 mL, 2.25 mmol) in DMF (2 mL). The resin was washed with DMF (104 mL) and deprotected with DMF/piperidine (4:1 v/v, 24 mL, 115 min, 110 min). After washing with DMF (104 mL) the last coupling was accomplished by shaking the resin for 90 min with a solution of Boc--Ala-OH (85 mg, 0.44 mmol), HATU (0.17 g, 0.44 mmol), HOAt (0.9 ml, 0.5 M in DMF) and DIPEA (0.25 mL, 1.43 mmol) in DMF (1 mL). After washing with DMF (104 mL) and DCM (104 mL) the peptide was cleaved off the resin by treating with TFA/HFIP/TIPS/H.sub.2O (1:94:2.5:2.5, v/v/v/v, 25 mL, 240 min). The resulting solutions were combined and the volume was reduced to a total of 5 mL by a stream of N.sub.2.

[0432] To the concentrated solution was added ice-cold diethyl ether. The formed suspension was centrifuged at 5000G at 0 C. for ten minutes and the supernatant was discarded, The residue was dissolved in 5 mL ACN/H.sub.2O/TFA (1:1:0.002 v/v/v) and it was purified by RP-HPLC to give 1i as a colorless, glassy solid after lyophilization. Yield: 80 mg, 67 mol (1TFA 3TFA salt), 30%. MS: m/z=972.56=[M+2TFA.sup.].sup.+, (calculated 972.56).

[0433] 1h (32 mg, 46 mol) was dissolved in DMF (1.14 mL) and 1i (47 mg, 53 mol) was added. To this mixture HATU (18 mg, 48 mol) and DIPEA (67 L, 0.38 mmol) were added and it was stirred for 90 min. The reaction was quenched by addition of AcOH (67 L) and water was added to a total volume of 5 mL. The resulting solution was purified by RP-HPLC to give 1j as colorless oil. Yield: 41 mg, 22 mol (1TFA 3TFA salt), 48%. MS: m/=769.97=[M+TFA.sup.].sup.2+, (calculated 769.89).

[0434] 1j (41 mg, 22 mol) was dissolved in TFA (1 mL) and stirred for 30 min before TFA was removed by a stream of N.sub.2. The residue was dissolved in H.sub.2O/ACN/TFA 1:1:0.002 (1.5 mL), frozen and lyophilized to give the free acid as colorless oil containing additional amounts of TFA. Yield: 63 mg, max. 22 mol (3TFA salt), quant. MS: m/z=685.03=[MH].sup.2+, (calculated 684.86). To a solution of the free acid (21 mg, 12 mol) in DMF (0.5 mL) was added Boc-1-tert-butoxy-1,2-dihydroisoquinoline (36 mg, 0.12 mmol). The mixture was stirred for 5 h before NHS (21 mg, 0.18 mmol) was added. The reaction was quenched by addition of AcOH (7 L) after 20 min. The mixture was cooled to 0 C. and was afterwards diluted with H.sub.2O/ACN/TFA 1:1:0.002 to a total volume of 2.5 mL. This solution was purified by RP-HPLC to give 1k as colorless oil. Yield: 11 mg, 5.2 mol (1TFA 3TFA salt), 43%. MS: m/z=790.37=[M+TFA.sup.].sup.2+, (calculated 790.37).

EXAMPLE 2

[0435] Synthesis of Purification Tag 2e

[0436] Purification tag 2e was synthesized according to the following scheme:

##STR00052## ##STR00053##

[0437] To a suspension of 6,6-dithiodinicotinic acid (0.62 g, 2 mmol) in ACN (20 mL) were added PyBOP (2.08 g, 4 mmol) and DIPEA (1.29 g, 1.74 mL, 10 mmol) and the mixture was stirred for 1 min. The obtained brown solution was added to a solution of 1,9-bis-Boc-1,5,9-triazanonane (1.99 g, 6 mmol) in a mixture of ACN (20 mL) and DMF (5 mL) and stirred for 2 h. The reaction mixture was diluted with EtOAc (150 mL) and the organic layer was washed with aq. HCl (10 mM, 5100 mL), saturated NaHCO.sub.3 solution (3100 mL) and brine (100 mL), subsequently. After drying over MgSO.sub.4 and filtration, the solvent was removed in vacuo and the crude residue was purified by flash chromatography to give 2a (1.92 g, max. 2 mmol) as a light yellow foam. The product contains a small, non-separable amount of tripyrrolidine phosphoramide, which is removed in the next step. MS: m/z=935.47=[M+H].sup.+, (calculated 935.47).

[0438] 2a (1.92 g, max. 2 mmol) was dissolved in TFA (10 mL) and the solution was stirred for 10 min. The reaction mixture was added dropwise to ice-cold diethyl ether (160 mL) to precipitate the product. The resulting suspension was centrifuged at 7000G and 2 C. for 3 min. The supernatant was discarded and the precipitate was dissolved in methanol (10 mL). This solution was added drop-wise to ice-cold diethyl ether (160 mL) and the formed suspension was centrifuged at 7000G and 2 C. for 3 min. After discarding the supernatant the precipitation procedure was accomplished two more times like described above. The remaining oily precipitate was dried in vacuo to give 2b (1.77 g, 1.45 mmol (6TFA salt), 73%) as a light brown, very hygroscopic powder. MS: m/z=535.26=[M+H].sup.+, (calculated 535.26).

[0439] To a solution of 2b (3.30 g, 2.7 mmol) in DMF (90 mL) were added DIPEA (5.4 mL, 31 mmol) and Boc-L-Lys(Boc)-OSu (5.62 g, 12.7 mmol). The mixture was stirred for 14 h before it was diluted with ethyl acetate (600 mL). The organic layer was washed with aq. HCl (10 mM, 5300 mL), sat. NaHCO.sub.3 solution (3300 mL) and brine (300 mL) and was dried over MgSO.sub.4. After filtration the solvent was removed in vacuo and the crude residue was purified by flash chromatography to give 2c (5.52 g, max. 2.7 mmol) as light yellow foam with 90% purity. MS: m/z=924.54=[M+2H].sup.2+, (calculated 924.53).

[0440] 2c (5.52 g, max. 2.7 mmol) was dissolved in TFA (20 mL). After stirring for 15 min the product was precipitated by adding the reaction mixture dropwise to ice-cold diethyl ether (160 mL). The resulting suspension was centrifuged at 7000G and 2 C. for 3 min. The supernatant was discarded and the precipitate was dissolved in methanol (10 mL). This solution was added dropwise to ice-cold diethyl ether (160 mL) and the formed suspension was centrifuged at 7000G and 2 C. for 3 min. After discarding the supernatant the precipitation procedure was accomplished two more times like described above. The remaining oily precipitate was dried in vacuo to give 2d (4.96 g, 2.27 mmol (10TFA salt), 84%) as a light brown, hygroscopic powder. MS: m/z=1046.64=[M+H].sup.+, (calculated 1046.64).

[0441] To a solution of 2d (1.53 g, 0.7 mmol) in dry DMF (20 mL) was added a solution of N,N-diemethylglycine (1.16 g, 11.2 mmol), PyBOP (5.83 g, 11.2 mmol) and DIPEA (3.23 g, 4.36 mL, 25 mmol) in DMF (35 mL) and stirred for 1 h. The mixture was then concentrated in vacuo to an approximate volume of 10 mL. To this residue water was added to a total volume of 100 mL and the solution was acidified to pH 1-2 by adding TFA. The turbid mixture was centrifuged at 5000G and 2 C. for 3 minutes. The oily precipitate was discarded and the supernatant was purified by RP-HPLC to give 2e (1.05 g, 0.37 mmol (10TFA salt), 53%) as a colorless oil. MS: m/z=864.54=[M+2H].sup.2+, (calculated 864.54).

EXAMPLE 3

[0442] Synthesis of Linker Reagent 3g

[0443] Linker reagent 3g was synthesized according to the following scheme:

##STR00054##

[0444] 2-Chlorotritylchloride resin (1.4 mmol/g, 1.43 g, 2 mmol) was weighted into a 20 ml syringe with frit. The resin was swollen twice with 10 mL DCM. N-Fmoc-N-methyl-L-Asp(OtBu)-OH (1.06 g, 2.5 mmol) was dissolved in DCM (6 mL) and drawn into the syringe. DIPEA (436 L, 2.5 mmol) was dissolved in DCM (1 mL) and drawn into the syringe. The syringe was agitated for 5 min. DIPEA (654 L, 3.75 mmol) was dissolved in DCM (1 mL) and drawn into the syringe. The syringe was agitated for 1 h. MeOH (2 mL) was drawn into the syringe and the syringe agitated for 30 min. The resin was washed 5 times with DMF (10 mL). The resin was agitated 3 times for 5 min with DMF:DBU:piperidine (96:2:2 v/v/v 7 mL). The resin was washed 5 times with DMF (5 mL). 6-Tritylmercaptohexanoic acid (1.95 g, 5 mmol) and PyBOP (2.6 g, 5 mmol) were dissolved in DMF (6 mL) and DIPEA (3.5 mL, 20 mmol) added. After 1 min preincubation the solution was drawn into the syringe and the syringe agitated for 3 h. The resin was washed 5 times with DMF (7 mL), 5 times with DCM (7 mL). A solution of HFIP/DCM ( v/v, 8 mL each) were drawn into the syringe and the syringe agitated 3 times for 30 min. The collected filtrates were concentrated in vacuo. Crude 3a (0.84 g, 1.45 mmol, 73%) was used without further purification in the next step. MS: m/z 598.18=[M+Na].sup.+, (calculated=598.26).

[0445] 3a (1.67 g, 2.9 mmol) was dissolved in DCM (20 mL) and N-Boc-N-methylethylenediamine (0.62 mL, 3.48 mmol) and PyBOP (1.81 g, 3.48 mmol) were added. DIPEA (2.02 mL, 11.6 mmol) was added and the reaction stirred for 1 h. AcOH (2 mL) was added, the mixture diluted with DCM (40 mL) and washed with water (220 mL). The organic layer was dried over MgSO.sub.4 and concentrated in vacuo and the crude residue was purified by flash chromatography to give 3b (1.74 g, 2.38 mmol, 82%). MS: m/z=754.19=[M+Na].sup.+, (calculated 754.39).

[0446] 3b (1.74 g, 2.38 mmol) and triphenylmethanol (0.62 g, 2.38 mmol) were dissolved in DCM (7.2 mL) and TFA (7.2 mL) was added with stirring. The reaction was stirred for 90 min and the solvents were removed in a stream of nitrogen over 45 min. The residue was co-evaporated with DCM. The residue was suspended in ACN/water/TFA (2:1:0.003 v/v/v, 14 mL) and filtered. The filtrate was purified by RP-HPLC to give 3c (0.9 g, 1.3 mmol TFA salt, 55%). MS: m/z 576.20=[M+H].sup.+, (calculated=576.29).

[0447] 3c (0.9 g, 1.3 mmol) was dissolved in DCM (20 mL) and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (0.46 g, 1.56 mmol) was added. DIPEA (0.45 mL, 2.6 mmol) was slowly added and the reaction stirred for 30 min. DIPEA (0.11 mL, 0.65 mmol) was added and the reaction stirred for 30 min. Again, DIPEA (0.11 mL, 0.65 mmol) was added and the reaction stirred for 60 min. AcOH (0.68 mL) was added and the mixture concentrated in vacuo and the crude residue was purified by flash chromatography to give 3d (1.04 g, max. 1.3 mmol). MS: m/z=754.28=[M+Na].sup.+, (calculated 754.28).

[0448] 3d (1.04 g, max. 1.3 mmol) was dissolved in HFIP/TES/water (39:1:1 v/v/v, 8.2 mL) and TFA (0.66 mL) was added. After stirring for 15 min the reaction was concentrated in vacuo, the residue suspended in ACN/water/TFA (1:1:0.002 v/v/v 12 mL) and filtered. The filtrate was purified by RP-HPLC to give 3e (0.32 g, 0.65 mmol, 50%). MS: m/z 490.19=[M+H].sup.+, (calculated=490.19).

[0449] 3e (0.18 g, 0.37 mmol) was dissolved in ACN/water/TFA (1:1:0.002 v/v/v, 3 mL). 2e (1.05 g, 0.37 mmol (10TFA salt) was dissolved in ACN/water (1:1 v/v, 20 mL). Both solutions were combined and pH 7.4 sodium phosphate (0.5 M, 4 mL) was added and the mixture stirred for 30 min. The pH of the solution was adjusted to ca. pH 2 by addition of ACN/water/TFA (1:1:0.22 v/v/v) and ACN was removed in vacuo. The residue was purified by RP-HPLC to give 3f (0.47 g, 0.24 mmol 5TFA salt, 65%). MS: m/z 676.86=[M+2H].sup.2+, (calculated=676.86).

[0450] 3f (0.18 g, 94 mol) was dissolved in ACN (6 mL) and NHS (92 mg, 0.8 mmol) and DCC (166 mg, 0.8 mmol) were added and the reaction stirred for 1 h. The solvent was removed in vacuo and the residue suspended in ACN/water/TFA (0.15:0.85:0.001 v/v/v, 6 mL) and filtered. The filtrate was purified by RP-HPLC to give 3g (129 mg, 64 mol 5TFA salt, 68%). MS: m/z 725.37=[M+H].sup.+, (calculated=725.37).

EXAMPLE 4

[0451] Synthesis and Purification of Transient Tagged Lucentis-Linker-Monoconjugate 4b

[0452] 120 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (3 mL of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 19 mg/mL. Linker reagent 1k was dissolved in DMSO to yield a concentration of 50 mM. 4 molar equivalents of linker reagent 1k relative to the amount of Lucentis were added to the Lucentis solution in 1 molar equivalent steps. The reaction mixture was mixed carefully after each linker reagent addition and incubated for 5 min at room temperature yielding a mixture of unmodified Lucentis, the protected, tagged Lucentis-linker monoconjugate 4a as well as protected, tagged Lucentis-linker bisconjugate.

[0453] The mixture of unmodified Lucentis, protected, tagged Lucentis-linker monoconjugate 4a and protected, tagged Lucentis-linker bisconjugate was buffer exchanged to 20 mM boric acid, pH 9.0 and incubated for 2 h at room temperature to remove the (5-methyl-2-oxo-1,3-dioxol-yl)-methyl oxocarbonyl protecting group of 4a yielding the tagged Lucentis-linker monoconjugate 4b.

[0454] 4b was purified from the reaction mixture by cationic ion exchange chromatography using an Aekta Purifier system equipped with a Source 15S 4.6/100 column. After loading of the reaction mixture (5-fold prediluted in mobile phase A) the following gradient of mobile phase A (20 mM MES, pH 5.7) and mobile phase B (20 mM MES, 500 mM NaCl, pH 5.7) was applied: linear increase from 3.2% B to 50% B in 20 column volumes leading to a separation of 4b from Lucentis and tagged Lucentis-linker bisconjugate (FIG. 1).

##STR00055## ##STR00056##

EXAMPLE 5

[0455] Deprotection of Transient Tagged Lucentis-Linker-Monoconjugate 4b Yielding Lucentis-Linker Monoconjugate 5a

[0456] Purified transient tagged Lucentis-linker-monoconjugate 4b was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and the protein concentration was adjusted to 1 mg/mL. The protein solution was cooled to 4 C. and 2 molar equivalents of 25 mM DTT in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 were added and incubated overnight at 4 C. yielding the Lucentis-linker monoconjugate 5a.

##STR00057##

EXAMPLE 6

[0457] Synthesis and Purification of Transient Tagged Lucentis-Linker Monoconjugate 6b

[0458] 400 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (10 mL of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 20.8 mg/mL. Linker reagent 3g was dissolved in DMSO to yield a concentration of 100 mM. 4.5 molar equivalents of linker reagent 3g relative to the amount of Lucentis were added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature yielding a mixture of unmodified Lucentis, the protected, tagged Lucentis-linker monoconjugate 6a and protected, tagged Lucentis-linker bisconjugate.

[0459] The mixture of Lucentis, protected, tagged Lucentis-linker monoconjugate 6a and protected, tagged Lucentis-linker bisconjugate was buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 6.5. To remove the (5-methyl-2-oxo-1,3-dioxol-yl)-methyl oxocarbonyl protecting group of 6a 0.5 M NH.sub.2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 6.5) was added to a final concentration of 45 mM and the deprotection reaction was incubated at room temperature for 2.5 h yielding the tagged Lucentis-linker monoconjugate 6b.

[0460] 6b was purified from the reaction mixture by cationic ion exchange chromatography using an Aekta Purifier system equipped with a Source 15S 6 mL column (FIG. 2). After loading of the reaction mixture (5-fold prediluted in mobile phase A) the following gradient of mobile phase A (20 mM MES, pH 5.7) and mobile phase B (20 mM MES, 500 mM NaCl, pH 5.7) was applied: linear increase from 0% B to 50% B in 20 column volumes leading to a separation of 6b from Lucentis and tagged Lucentis-linker bisconjugate.

##STR00058## ##STR00059##

EXAMPLE 7

[0461] Deprotection of Transient Tagged Lucentis-Linker-Monoconjugate 6b Yielding Lucentis-Linker Monoconjugate 7a

[0462] Purified transient tagged Lucentis-linker-monoconjugate 6b was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and the protein concentration was adjusted to 10 mg/mL. The protein solution was cooled to 4 C. and 5 molar equivalents of 25 mM DTT in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 were added and incubated overnight at 4 C. yielding the Lucentis-linker monoconjugate 7a.

##STR00060##

Abbreviations

[0463] ACN acetonitrile [0464] AcOH acetic acid [0465] Ado 8-amino-3,6-dioxaoctanoic acid [0466] aq. aqueous [0467] Asp aspartate [0468] -Ala beta-alanine [0469] Boc tert-butyloxycarbonyl [0470] CIEC cationic ion exchange chromatography [0471] cv column volume [0472] DBU 1,8-diazabicyclo (5.4.0)undec-7-ene [0473] DCC dicyclohexylcarbodiimide [0474] DCM dichloromethane [0475] DIPEA diisopropylethylamine [0476] DMF dimethylformamide [0477] DTT dithiothreitol [0478] EDTA ethylendiaminetetraacetic acid [0479] Fmoc fluorenylmethyloxycarbonyl [0480] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0481] HFIP hexafluoroisopropanol [0482] HOAt 1-hydroxy-7-azabenzotriazole [0483] HOBt 1-hydroxybenzotriazole [0484] HPLC high performance liquid chromatography [0485] Lys lysine [0486] max. maximal [0487] Me methyl [0488] MeOH methanol [0489] MES 2-(N-morpholino)ethanesulfonic acid [0490] MS mass spectrometry [0491] NHS N-hydroxysuccinimide [0492] PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0493] RP-HPLC reversed phasehigh performance liquid chromatography [0494] sat. saturated [0495] Su N-hydroxusuccinimidyl [0496] tBu and t-Bu tert.-butyl [0497] TES triethylsilane [0498] TFA trifluoroacetic acid [0499] THF tetrahydrofurane [0500] TLC thin layer chromatography [0501] Trt trityl [0502] TBTU N,N,NN-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate