U.S. patent application number 17/667002 was filed with the patent office on 2022-08-25 for methods for antibody drug conjugation, purification, and formulation.
The applicant listed for this patent is ImmunoGen, Inc.. Invention is credited to Robert W. HERBST, Benjamin M. HUTCHINS, Daniel F. MILANO, Michael R. REARDON, Richard A. SILVA.
Application Number | 20220267371 17/667002 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220267371 |
Kind Code |
A1 |
MILANO; Daniel F. ; et
al. |
August 25, 2022 |
METHODS FOR ANTIBODY DRUG CONJUGATION, PURIFICATION, AND
FORMULATION
Abstract
Methods of producing, purifying, and formulating antibody drug
conjugates (ADCs) are provided herein. The methods use continuous
conjugation processes, single-pass tangential flow filtration,
countercurrent diafiltration, and/or in-line process automation
technologies. The methods decrease process times and costs and
improve product consistency.
Inventors: |
MILANO; Daniel F.; (Reading,
MA) ; REARDON; Michael R.; (North Attleboro, MA)
; SILVA; Richard A.; (Needham, MA) ; HUTCHINS;
Benjamin M.; (Boxborough, MA) ; HERBST; Robert
W.; (Braintree, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ImmunoGen, Inc. |
Weltham |
MA |
US |
|
|
Appl. No.: |
17/667002 |
Filed: |
February 8, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16245890 |
Jan 11, 2019 |
11274121 |
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17667002 |
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62617021 |
Jan 12, 2018 |
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62673535 |
May 18, 2018 |
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International
Class: |
C07K 1/34 20060101
C07K001/34; A61K 47/68 20060101 A61K047/68; A61K 31/5383 20060101
A61K031/5383; A61K 31/5517 20060101 A61K031/5517; C07K 1/16
20060101 C07K001/16; G01N 33/68 20060101 G01N033/68; C07K 16/00
20060101 C07K016/00 |
Claims
1-28. (canceled)
29. A method for concentrating an antibody drug conjugate (ADC),
purifying an ADC, or transferring an ADC to a formulation buffer
comprising using single-pass tangential flow filtration and/or
countercurrent diafiltration.
30-31. (canceled)
32. The method of claim 29, wherein the ADC was produced in a batch
conjugation process.
33. The method of claim 29, wherein the ADC was produced in a
continuous conjugation process.
34. The method of claim 29, and wherein the single-pass tangential
flow filtration uses an ultrafiltration membrane.
35. The method of claim 29, wherein the single-pass tangential flow
filtration uses a diafiltration membrane.
36. The method of claim 29, wherein the method improves the
consistency of the ADC production.
37. The method of claim 29, wherein the method decreases the time
for ADC production.
38. The method of claim 29, wherein the single-pass tangential flow
filtration and/or the countercurrent diafiltration improves the
consistency of the ADC production.
39. The method of claim 29, wherein the single-pass tangential flow
filtration and/or the countercurrent diafiltration decreases the
time for ADC concentration, purification, or transfer.
40. The method of claim 29, wherein the single-pass tangential flow
filtration and/or the countercurrent diafiltration decreases the
amount of buffer used.
41. The method of claim 29, wherein the method further comprises
in-line monitoring of an analyte.
42. A method for producing an antibody drug conjugate (ADC) wherein
(i) in-line monitoring is used to measure the addition of a
component to an ADC conjugation reaction, (ii) in-line monitoring
of an analyte is used to determine when to stop adding conjugation
buffer to an ADC conjugation reaction (iii) in-line monitoring of
an analyte is used to determine when to stop recirculating
conjugation buffer in an ADC conjugation reaction, (iv) in-line
monitoring of an analyte is used to determine when to start rinsing
conjugation buffer from an ADC conjugation reaction, and/or (v)
in-line monitoring of an analyte is used to determine when to stop
adding a conjugation reaction component to an ADC conjugation
reaction.
43-49. (canceled)
50. A method for purifying an antibody drug conjugate (ADC) after
an ADC conjugation process wherein the method comprises in-line
monitoring of an analyte.
51.-77. (canceled)
78. The method of claim 29, wherein the ADC comprises an antibody
or antigen-binding fragment that specifically binds to CD37, CD33,
FOLR1, CD123, CD19, cMET, ADAM9, or HER2.
79.-83. (canceled)
84. The method of claim 29, wherein the ADC comprises a linker
selected from the group consisting of SMCC, sSPDB, and a peptide
linker.
85. The method of claim 29, wherein the ADC comprises a
maytansinoid or an indolino-benzodiazepine.
86-87. (canceled)
88. The method of claim 29, wherein the ADC is IMGN529, IMGN779,
IMGN853, IMGN632, or Kadcyla.
89.-92. (canceled)
93. A method of producing an ADC comprising (i) increasing a first
temperature of a conjugation process by at least 5.degree. C. to an
elevated temperature, (ii) decreasing a first temperature of
conjugation process by at least 5.degree. C. to a reduced
temperature, (iii) changing a first pH of a conjugation process by
at least 0.5 to an altered pH, wherein the elevated temperature,
the reduced temperature, or the altered pH is maintained for no
more than 20 minutes.
94.-139. (canceled)
140. An ADC produced according to the method of claim 29.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 16/245,890 (now allowed), filed Jan. 11, 2019,
which claims the benefit of U.S. Provisional Appl. Nos. 62/673,535,
filed May 18, 2018 and 62/617,021, filed Jan. 12, 2018, each of
which is herein incorporated by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence
listing, file name: Sequence-Listing-2921-0990003.txt; size: 41,229
bytes; and date of creation: Feb. 4, 2022, filed herewith, is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The field of the invention generally relates to methods of
producing, purifying, and formulating antibody drug conjugates
(ADCs) using continuous conjugation processes, single-pass
tangential flow filtration, countercurrent diafiltration, and/or
in-line process automation technologies.
BACKGROUND
[0004] A manufacturing bottleneck associated with antibody drug
conjugate (ADC) conjugation is removal of impurities and exchange
of the ADC into a stable buffer. This is currently achieved using
bind and elute column chromatography or bulk (conventional)
diafiltration by tangential flow filtration (TFF). Bulk
diafiltration involves priming the TFF system with buffer A (i.e.,
the buffer that the conjugate is initially in). The ADC (in buffer
A) is then added to a retentate vessel where it mixes with the
prime volume. A feed pump for the retentate vessel pumps the
conjugate from the retentate over the TFF membrane where it is
either retained (and returns to the retentate) or discarded to
waste. Another pump (the diafiltration pump) would feed from the
vessel to buffer B contained in a separate vessel (i.e., the buffer
into which the ADC will be exchanged) with a feed line from the
vessel into the retentate vessel. Both pumps are started and the
conjugate in the retentate vessel begins passing over the TFF
membrane. As buffer is removed via the waste stream, the volume in
the retentate is maintained by adding Buffer B (in equal volume) to
the retentate vessel. As a result, the conjugate slowly is
exchanged into Buffer B. This purification method is costly and not
optimal for large-scale manufacturing because of the requirement to
recirculate the ADC over the TFF membranes for multiple cycles. The
recirculation results in high processing volumes and times as well
as increased exposure of the ADC to potentially high shear zones.
ADC manufacturing using bulk processing takes about 4-5 days to
produce a batch of purified, formulated conjugate, often times at
low yields. Accordingly, improved ADC conjugation processes are
needed.
BRIEF SUMMARY OF THE INVENTION
[0005] Single-pass tangential flow filtration (SPTFF) systems such
as those available from Pall Life Sciences are used in
bioprocessing platforms, often as a way to reduce volume or
concentrate biomolecules such as antibodies. However, the present
inventors have surprisingly discovered that SPTFF can be linked to
antibody drug conjugation reactions to facilitate, not only the
concentration of antibody drug conjugates (ADCs), but also to
remove unconjugated products from the conjugation reaction and
exchange the purified ADC into a formulation buffer, thereby
enabling an entirely continuous ADC production and processing
method. The present inventors have also discovered that
countercurrent diafilitration can also be used in antibody drug
conjugation reactions for similar purposes.
[0006] In some embodiments, a continuous method for producing an
antibody drug conjugate (ADC) composition comprises (i) conjugating
an antibody or antigen-binding fragment thereof to a drug to form
an ADC, (ii) removing unconjugated drug, and (iii) exchanging the
ADC into a stable buffer, wherein (i) to (iii) are performed
continuously, and wherein single-pass tangential flow filtration
(SPTFF) is used to remove the unconjugated drug and/or exchange the
ADC into the stable buffer.
[0007] In some embodiments, SPTFF is used to remove the
unconjugated drug to exchange the ADC into the stable buffer. In
some embodiments, flow-through column chromatography is used to
remove the unconjugated drug and SPTFF is used to exchange the ADC
into the stable buffer.
[0008] In some embodiments, a continuous method for producing an
antibody drug conjugate (ADC) composition comprises (i) conjugating
an antibody or antigen-binding fragment thereof to a drug to form
an ADC, (ii) removing unconjugated drug, and (iii) exchanging the
ADC into a stable buffer, wherein (i) to (iii) are performed
continuously, and wherein countercurrent diafilitration is used to
remove the unconjugated drug and/or exchange the ADC into the
stable buffer.
[0009] In some embodiments, countercurrent diafilitration is used
to remove the unconjugated drug to exchange the ADC into the stable
buffer. In some embodiments, flow-through column chromatography is
used to remove the unconjugated drug and countercurrent
diafilitration is used to exchange the ADC into the stable
buffer.
[0010] In some embodiments, the method further comprises
pre-processing the antibody or antigen-binding fragment thereof. In
some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed continuously with (i)
to (iii). In some embodiments, the pre-processing of the antibody
or antigen-binding fragment thereof is performed using SPTFF. In
some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed using countercurrent
diafilitration. In some embodiments, only one SPTFF or
countercurrent diafiltration step is used in the pre-processing. In
some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed in bulk prior to (i)
to (iii).
[0011] In some embodiments, the method further comprises
pre-processing the drug for conjugation.
[0012] In some embodiments, a continuous method for producing an
antibody drug conjugate (ADC) composition comprises (i)
pre-processing an antibody or antigen-binding fragment thereof,
(ii) conjugating the antibody or antigen-binding fragment thereof
to a drug to form an ADC, (iii) removing unconjugated drug, and
(iv) exchanging the ADC into a stable buffer, wherein (i) to (iv)
are performed continuously, and wherein single-pass tangential flow
filtration (SPTFF) is used to remove the unconjugated drug and/or
exchange the ADC into the stable buffer.
[0013] In some embodiments, a continuous method for producing an
antibody drug conjugate (ADC) composition comprises (i)
pre-processing an antibody or antigen-binding fragment thereof,
(ii) conjugating the antibody or antigen-binding fragment thereof
to a drug to form an ADC, (iii) removing unconjugated drug, and
(iv) exchanging the ADC into a stable buffer, wherein (i) to (iv)
are performed continuously, and wherein countercurrent
diafiltration is used to remove the unconjugated drug and/or
exchange the ADC into the stable buffer.
[0014] In some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof comprises exchanging the antibody
or antigen-binding fragment thereof into a buffer for conjugation.
In some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof comprises reducing the antibody or
antigen-binding fragment thereof and/or oxidizing the antibody or
antigen-binding fragment thereof. In some embodiments, the reducing
and/or the oxidizing are achieved without using SPTFF or
countercurrent diafiltration. In some embodiments, the reducing
and/or the oxidizing are achieved using only one SPTFF or
countercurrent diafiltration step. In some embodiments, the
reducing and/or the oxidizing are achieved using more than one
SPTFF or countercurrent diafiltration step.
[0015] In some embodiments, a method for producing an antibody drug
conjugate (ADC) in a continuous conjugation process comprises
adding one or more conjugation reaction reagents to a conjugation
reaction while the conjugation reaction proceeds and after at least
one conjugation reaction product (ADC) has formed. In some
embodiments, the conjugation reaction reagent comprises an antibody
or antigen-binding fragment thereof. In some embodiments, the
conjugation reaction reagent comprises a drug attached to a linker.
In some embodiments, the conjugation reaction reagent comprises a
linker. In some embodiment, the conjugation reaction reagent
comprises an antibody or antigen-binding fragment thereof attached
to a linker. In some embodiments, the conjugation reaction reagent
comprises a drug.
[0016] In some embodiments, the conjugation reaction occurs in a
flow reactor.
[0017] In some embodiments, the method further comprises
pre-processing an antibody or antigen-binding fragment thereof. In
some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed continuously with the
conjugation reaction. In some embodiments, the pre-processing of
the antibody or antigen-binding fragment thereof is performed using
SPTFF. In some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed using countercurrent
diafiltration. In some embodiments, only one SPTFF or
countercurrent diafiltration step is used in the pre-processing. In
some embodiments, the pre-processing of the antibody or
antigen-binding fragment thereof is performed in bulk prior to the
conjugation reaction. In some embodiments, the pre-processing of
the antibody or antigen-binding fragment thereof comprises
exchanging the antibody or antigen-binding fragment thereof into a
buffer for conjugation. In some embodiments, the pre-processing of
the antibody or antigen-binding fragment thereof comprises reducing
the antibody or antigen-binding fragment thereof and/or oxidizing
the antibody or antigen-binding fragment thereof.
[0018] In some embodiments, the ADC is concentrated using
single-pass tangential flow filtration. In some embodiments, the
ADC is purified using single-pass tangential flow filtration. In
some embodiments, the ADC is transferred to a formulation buffer
using single-pass tangential flow filtration.
[0019] In some embodiments, the ADC is concentrated using
countercurrent diafiltration. In some embodiments, the ADC is
purified using countercurrent diafiltration. In some embodiments,
the ADC is transferred to a formulation buffer using countercurrent
diafiltration.
[0020] In some embodiments, the ADC is concentrated, purified,
and/or transferred to a formulation buffer using flow-through
chromatography.
[0021] In some embodiments, a method for concentrating an antibody
drug conjugate (ADC) comprises using single-pass tangential flow
filtration. In some embodiments, a method for purifying an antibody
drug conjugate (ADC) comprises using single-pass tangential flow
filtration. In some embodiments, a method for transferring an
antibody drug conjugate (ADC) to a formulation buffer comprises
using single-pass tangential flow filtration. In some embodiments,
a method for concentrating an antibody drug conjugate (ADC)
comprises using countercurrent diafiltration. In some embodiments,
a method for purifying an antibody drug conjugate (ADC) comprises
using countercurrent diafiltration. In some embodiments, a method
for transferring an antibody drug conjugate (ADC) to a formulation
buffer comprises using countercurrent diafiltration. In some
embodiments, the ADC was produced in a batch conjugation process.
In some embodiments, the ADC was produced in a continuous
conjugation process.
[0022] In some embodiments, the single-pass tangential flow
filtration uses an ultrafiltration membrane. In some embodiments,
the single-pass tangential flow filtration uses a diafiltration
membrane.
[0023] In some embodiments, the method improves the consistency of
the ADC production. In some embodiments, the method decreases the
time for ADC production.
[0024] In some embodiments, the single-pass tangential flow
filtration improves the consistency of the ADC production. In some
embodiments, the single-pass tangential flow filtration decreases
the time for ADC concentration, purification, or transfer. In some
embodiments, the single-pass tangential flow filtration decreases
the amount of buffer used.
[0025] In some embodiments, the countercurrent diafiltration
improves the consistency of the ADC production. In some
embodiments, the countercurrent diafiltration decreases the time
for ADC concentration, purification, or transfer. In some
embodiments, the countercurrent diafiltration decreases the amount
of buffer used.
[0026] In some embodiments, the method further comprises in-line
monitoring of an analyte.
[0027] In some embodiments, a method for producing an antibody drug
conjugate (ADC) comprising using in-line monitoring to measure the
addition of a component to an ADC conjugation reaction.
[0028] In some embodiments, the in-line monitoring measures the
concentration of a component added to the ADC conjugation
reaction.
[0029] In some embodiments, the in-line monitoring measures the
flow rate of a component added to the ADC conjugation reaction. In
some embodiments, the component is an antibody or antigen-binding
fragment thereof, a drug, a linker, drug attached to a linker, an
antibody or antigen-binding fragment thereof attached to a linker,
and/or a conjugation buffer.
[0030] In some embodiments, the method further comprises in-line
monitoring to measure the addition of a component to an in-situ
reaction. In some embodiments, the component is a drug, a linker,
and/or an in-situ reaction buffer.
[0031] In some embodiments, a method for producing an antibody drug
conjugate (ADC) comprises in-line monitoring of an analyte to
determine when (i) to stop adding conjugation buffer to an ADC
conjugation reaction (ii) to stop recirculating conjugation buffer
in an ADC conjugation reaction, and/or (iii) to start rinsing
conjugation buffer from an ADC conjugation reaction. In some
embodiments, the analyte is unconjugated drug or unconjugated drug
attached to linker.
[0032] In some embodiments, a method for purifying an antibody drug
conjugate (ADC) after an ADC conjugation process comprises in-line
monitoring of an analyte. In some embodiments, the purification
comprises filtration. In some embodiments, the filtration is
ultrafiltration or diafiltration. In some embodiments, the
filtration is tangential flow filtration. In some embodiments, the
tangential flow filtration is single-pass tangential flow
filtration. In some embodiments, the filtration is countercurrent
diafiltration.
[0033] In some embodiments, the analyte is in the retentate. In
some embodiments, the analyte is the concentration of unconjugated
drug or unconjugated drug attached to linker. In some embodiments,
the analyte is the concentration of an ADC or an antibody or
antigen-binding fragment thereof. In some embodiments, the analyte
is the concentration of a component of a conjugation reaction
buffer or a filtration buffer.
[0034] In some embodiments, the analyte is the pH.
[0035] In some embodiments, the analyte is in the permeate. In some
embodiments, the analyte is the concentration of an ADC or an
antibody or antigen-binding fragment thereof. In some embodiments,
the analyte is the concentration of unconjugated drug or
unconjugated drug attached to linker.
[0036] In some embodiments, the purification comprises
chromatography, and the analyte is measured at the end of a
chromatography column. In some embodiments, the analyte is an ADC
or an antibody or antigen-binding fragment thereof. In some
embodiments, the analyte is unconjugated drug, unconjugated drug
attached to linker, a component of a conjugation reaction buffer,
or a component of a chromatography buffer.
[0037] In some embodiments, the conjugation reaction is a batch
conjugation reaction. In some embodiments, the conjugation reaction
is a continuous conjugation reaction.
[0038] In some embodiments, the in-line monitoring is performed
using variable path-length technology, a flow cell device, a UV
sensor, Raman spectroscopy, or Fourier Transform Infrared
spectroscopy (FITR).
[0039] In some embodiments, the in-line monitoring of the analyte
is performed using a FlowVPE.
[0040] In some embodiments, the in-line monitoring improves ADC
yield. In some embodiments, the in-line monitoring improves ADC
recovery. In some embodiments, the in-line monitoring decreases
time for ADC production. In some embodiments, the in-line
monitoring decreases the amount of buffer used.
[0041] In some embodiments, a method for producing an antibody drug
conjugate (ADC) comprises in-line monitoring of an analyte to
determine when to stop adding a conjugation reaction component to
an ADC conjugation reaction. In some embodiments, the component is
an antibody or antigen-binding fragment thereof, a drug, a linker,
a drug attached to a linker, and/or a conjugation reaction
buffer.
[0042] In some embodiments, the ADC comprises an antibody. In some
embodiments, wherein the ADC comprises an antigen-binding fragment
of an antibody. In some embodiments, the ADC comprises an antibody
or antigen-binding fragment that specifically binds to CD37, CD33,
FOLR1, CD123, CD19, cMET, ADAM9, or HER2. In some embodiments, the
antibody or antigen-binding fragment thereof comprises the VH CDR1,
VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of huMov19, Z4681A,
or G4732A. In some embodiments, the CDRs are the Kabat-defined
CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs. In some
embodiments, the antibody or antigen-binding fragment thereof
comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3
sequences comprising the sequences of SEQ ID NOs:1-6, respectively,
SEQ ID NOs:7-12, respectively, SEQ ID NOs:13-18, respectively, SEQ
ID NOs:19-24, respectively, SEQ ID NOs:25-30, or respectively, SEQ
ID NOs:31-36, respectively. In some embodiments, the antibody or
antigen-binding fragment thereof comprises VH and VL sequences
comprising the sequence of SEQ ID NOs:37 and 38, respectively, SEQ
ID NOs:37 and 39, respectively, SEQ ID NOs:40 and 41, respectively,
SEQ ID NOs:42 and 43, respectively, SEQ ID NOs:44 and 45,
respectively, SEQ ID NOs: 46 and 47, respectively, or SEQ ID NOs:48
and 49, respectively. In some embodiments, the antibody comprises
heavy and light chain sequences comprising the sequences of SEQ ID
NOs:50 and 51, respectively, SEQ ID NOs:50 and 52, respectively,
SEQ ID NOs:53 and 54, respectively, or SEQ ID NOs:55 and 56,
respectively.
[0043] In some embodiments, the ADC comprises a linker selected
from the group consisting of SMCC, sSPDB, and a peptide linker. In
some embodiments, the ADC comprises a maytansinoid or an
indolino-benzodiazepine. In some embodiments, the maytansinoid is
DM1 or DM4. In some embodiments, the indolino-benzodiazepine is
DGN462 or DGN549.
[0044] In some embodiments, the ADC is IMGN529, IMGN779, IMGN853,
IMGN632, or Kadcyla.
[0045] In some embodiments, a method for producing IMGN853
comprises (i) mixing huMov19 antibody and DM4-linked to sulfo-SPDB
in a continuous conjugation process to form IMGN853, optionally
wherein in-line monitoring is used to measure the concentration of
huMov19 antibody added to the conjugation reaction, (ii) optionally
concentrating IMGN853 using single-pass tangential flow filtration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, (iii)
purifying IMGN853 using single-pass tangential flow filtration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, and (iv)
exchanging the IMGN853 into a formulation buffer using single-pass
tangential flow filtration, optionally wherein in-line monitoring
is used to measure the concentration of impurities in the
retentate.
[0046] In some embodiments, a method for producing IMGN853
comprises (i) mixing huMov19 antibody and DM4-linked to sulfo-SPDB
in a continuous conjugation process to form IMGN853, optionally
wherein in-line monitoring is used to measure the concentration of
huMov19 antibody added to the conjugation reaction, (ii) optionally
concentrating IMGN853 using single-pass tangential flow filtration
and/or countercurrent diafiltration, optionally wherein in-line
monitoring is used to measure the concentration of unconjugated
drug in the retentate, (iii) purifying IMGN853 using single-pass
tangential flow filtration and/or countercurrent diafiltration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, and (iv)
exchanging the IMGN853 into a formulation buffer using single-pass
tangential flow filtration and/or countercurrent diafiltration,
optionally wherein in-line monitoring is used to measure the
concentration of impurities in the retentate.
[0047] In some embodiments, a method for producing IMGN779
comprises (i) mixing Z4681A antibody and DGN462-linked to
sulfo-SPDB in a continuous conjugation process to form IMGN779,
optionally wherein in-line monitoring is used to measure the
concentration of Z4681A antibody added to the conjugation reaction,
(ii) optionally concentrating IMGN779 using single-pass tangential
flow filtration, optionally wherein in-line monitoring is used to
measure the concentration of unconjugated drug in the retentate,
(iii) purifying IMGN779 using single-pass tangential flow
filtration, optionally wherein in-line monitoring is used to
measure the concentration of unconjugated drug in the retentate,
and (iv) exchanging the IMGN779 into a formulation buffer using
single-pass tangential flow filtration, optionally wherein in-line
monitoring is used to measure the concentration of impurities in
the retentate.
[0048] In some embodiments, a method for producing IMGN779
comprises (i) mixing Z4681A antibody and DGN462-linked to
sulfo-SPDB in a continuous conjugation process to form IMGN779,
optionally wherein in-line monitoring is used to measure the
concentration of Z4681A antibody added to the conjugation reaction,
(ii) optionally concentrating IMGN779 using single-pass tangential
flow filtration and/or countercurrent diafiltration, optionally
wherein in-line monitoring is used to measure the concentration of
unconjugated drug in the retentate, (iii) purifying IMGN779 using
single-pass tangential flow filtration and/or countercurrent
diafiltration, optionally wherein in-line monitoring is used to
measure the concentration of unconjugated drug in the retentate,
and (iv) exchanging the IMGN779 into a formulation buffer using
single-pass tangential flow filtration and/or countercurrent
diafiltration, optionally wherein in-line monitoring is used to
measure the concentration of impurities in the retentate.
[0049] In some embodiments, a method for producing IMGN632
comprises (i) mixing G4732A antibody and sulfonated DNG549C in a
continuous conjugation process to form IMGN632, (ii) optionally
concentrating IMGN632 using single-pass tangential flow filtration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, (iii)
purifying IMGN632 using single-pass tangential flow filtration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, and (iv)
exchanging the IMGN632 into a formulation buffer using single-pass
tangential flow filtration, optionally wherein in-line monitoring
is used to measure the concentration of impurities in the
retentate. In some embodiments, the G4732A antibody is reduced and
oxidized in only one SPTFF step prior the conjugation process.
[0050] In some embodiments, a method for producing IMGN632
comprises (i) mixing G4732A antibody and sulfonated DNG549C in a
continuous conjugation process to form IMGN632, (ii) optionally
concentrating IMGN632 using single-pass tangential flow filtration
and/or countercurrent diafiltration, optionally wherein in-line
monitoring is used to measure the concentration of unconjugated
drug in the retentate, (iii) purifying IMGN632 using single-pass
tangential flow filtration and/or countercurrent diafiltration,
optionally wherein in-line monitoring is used to measure the
concentration of unconjugated drug in the retentate, and (iv)
exchanging the IMGN632 into a formulation buffer using single-pass
tangential flow filtration and/or countercurrent diafiltration,
optionally wherein in-line monitoring is used to measure the
concentration of impurities in the retentate. In some embodiments,
the G4732A antibody is reduced and oxidized in only one SPTFF or
countercurrent diafiltration step prior the conjugation
process.
[0051] In some embodiments, the method comprises increasing a first
temperature of a conjugation process by at least 5.degree. C. to an
elevated temperature, wherein the elevated temperature is
maintained for no more than 20 minutes.
[0052] In some embodiments, a method of producing an ADC comprises
increasing a first temperature of a conjugation process by at least
5.degree. C. to an elevated temperature, wherein the elevated
temperature is maintained for no more than 20 minutes.
[0053] In some embodiments, the elevated temperature is no more
than 55.degree. C. In some embodiments, the first temperature is
increased by at least 10.degree. C., by at least 15.degree. C., by
at least 20.degree. C., or by at least 30.degree. C.
[0054] In some embodiments, the elevated temperature is 35.degree.
C. to 55.degree. C. or is 40.degree. C. to 50.degree. C.
[0055] In some embodiments, increasing the first temperature to the
elevated temperature does not take longer than 2 minutes or does
not take longer than 1 minute.
[0056] In some embodiments, the method further comprises decreasing
the elevated temperature, optionally to the first temperature. In
some embodiments, decreasing the elevated temperature, optionally
to the first temperature, does not take longer than 2 minutes or
does not take longer than 1 minute.
[0057] In some embodiments, the steps of increasing the first
temperature to the elevated temperature and then decreasing the
elevated temperature are repeated at least twice or at least three
times. In some embodiments, the steps of increasing the first
temperature to the elevated temperature and then decreasing the
elevated temperature are repeated 2-20 times or 5-10 times.
[0058] In some embodiments, the method comprises decreasing a first
temperature of a conjugation process by at least 5.degree. C. to a
reduced temperature, wherein the reduced temperature is maintained
for no more than 20 minutes.
[0059] In some embodiments, a method of producing an ADC comprises
decreasing a first temperature of a conjugation process by at least
5.degree. C. to a reduced temperature, wherein the reduced
temperature is maintained for no more than 20 minutes. In some
embodiments, the first temperature is decreased by no more than
30.degree. C. In some embodiments, the temperature is decreased by
at least 10.degree. C., by at least 15.degree. C., or by at least
20.degree. C.
[0060] In some embodiments, decreasing the first temperature to the
reduced temperature does not take longer than 2 minutes or does not
take longer than 1 minute.
[0061] In some embodiments, the method further comprises increasing
the reduced temperature, optionally to the first temperature. In
some embodiments, increasing the reduced temperature, optionally to
the first temperature, does not take longer than 2 minutes or does
not take longer than 1 minute.
[0062] In some embodiments, the steps of decreasing the first
temperature to the reduced temperature and then increasing the
reduced temperature are repeated at least twice or at least three
times. In some embodiments, the steps of decreasing the first
temperature to the reduced temperature and then increasing the
reduced temperature are repeated 2-20 times or 5-10 times.
[0063] In some embodiments, wherein the elevated temperature or
reduced temperature is maintained for no more than 15 minutes. In
some embodiments, the elevated temperature or reduced temperature
is maintained for 30 seconds to 15 minutes. In some embodiments,
the elevated temperature or reduced temperature is maintained for
15 to 20 minutes. In some embodiments, the elevated temperature or
reduced temperature is maintained for 10 to 15 minutes. In some
embodiments, the elevated temperature or reduced temperature is
maintained for 5 to 10 minutes. In some embodiments, the elevated
temperature or reduced temperature is maintained for 1 to 5
minutes.
[0064] In some embodiments, the method comprises changing a first
pH of a conjugation process by at least 0.5 to an altered pH,
wherein the altered pH is maintained for no more than 20
minutes.
[0065] In some embodiments, a method of producing an ADC comprises
changing a first pH of a conjugation process by at least 0.5 to an
altered pH, wherein the altered pH is maintained for no more than
20 minutes.
[0066] In some embodiments, the first pH is increased by at least
1, optionally wherein the altered pH does not exceed 9. In some
embodiments, the first pH is increased by at least 2 or by at least
3.
[0067] In some embodiments, the first pH is decreased by at least
1, optionally wherein the altered pH does not go below 4. In some
embodiments, the first pH is decreased by at least 2 or by at least
3.
[0068] In some embodiments, the method further comprises changing
the altered pH, optionally to the first pH. In some embodiments,
the steps of changing the first pH to the altered pH and then
changing the altered pH are repeated at least twice or at least
three times. In some embodiments, the steps of changing the first
pH to the altered pH and then changing the altered pH are repeated
2-20 times or 5-10 times.
[0069] In some embodiments, the altered pH is maintained for no
more than 15 minutes. In some embodiments, the altered pH is
maintained for 30 seconds to 15 minutes. In some embodiments, the
altered pH is maintained for 15 to 20 minutes. In some embodiments,
the altered pH is maintained for 10 to 15 minutes. In some
embodiments, the altered pH is maintained for 5 to 10 minutes. In
some embodiments, the altered pH is maintained for 1 to 5
minutes.
[0070] Also provided herein are ADCs produced according to the
methods provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0071] FIG. 1 shows a fully continuous antibody drug conjugate
(ADC) manufacturing process with integrated feedback control
mechanisms (bottom) and its relation to conventional batch
processing (top).
[0072] FIG. 2 is a flow diagram showing a semi-batch operation,
incorporating continuous-enabling technologies (bottom) and its
relation to conventional batch processing (top).
[0073] FIG. 3 is a flow diagram showing the coupling of semi-batch
unit operations (bottom) and its relation to conventional batch
processing (top).
[0074] FIG. 4 is a flow diagram showing the continuous conjugation
process for the conjugation of the antibody drug conjugate ADC
"IMGN853" described in Example 1. "mAb" refers to monoclonal
antibody (i.e., huMov19). "D" refers to drug (i.e., the
maytansinoid DM4), and "L" refers to linker (i.e., sSPDB.)
[0075] FIG. 5 shows the setup for continuous conjugation studies
described in Example 1A. The first syringe pump (L/D/DMA) feeds the
in-situ components dissolved in DMA. The Buffer (IS) syringe pump
feeds the in-situ reaction buffer (same as conjugation reaction
buffer, but pH is 7.6 for in-situ reaction). The third syringe
feeds the antibody and pH 8.7 conjugation reaction buffer. "mAb"
refers to monoclonal antibody (i.e., huMov19). "D" refers to drug
(i.e., the maytansinoid DM4), and "L" refers to linker (i.e.,
sSPDB. "IS" refers to in-situ.
[0076] FIG. 6 shows free drug impurity levels for the four main
impurity species in samples taken at the ILC, ILDF1 and ILDF2
stages described in Example 1B.
[0077] FIG. 7 shows free drug impurity levels for the four main
impurity species in samples taken at ILDF1 at 15 mg/mL feed
concentration as described in Example 1B.
[0078] FIG. 8 shows a comparison of free drug impurity levels for
continuous and batch TFF purification processes. The continuous
(striped bars) conjugate material was processed at 15 g/L starting
concentration whereas the batch (solid bars) material was processed
at 30 g/L as described in Example 1B.
[0079] FIG. 9 shows free drug impurity levels for the four main
impurity species in samples taken at ILDF1 at 30 mg/mL feed
concentration as described in Example 1B.
[0080] FIG. 10 is a flow diagram showing the continuous conjugation
process for the conjugation of the antibody drug conjugate (ADC)
"IMGN632" described in Example 2. "mAb" refers to monoclonal
antibody (i.e., G4723A).
[0081] FIG. 11 shows the chemical structure for IMGN632. IMGN632 is
a composition comprising ADCs containing the anti-CD123 G4723
antibody linked to the cytotoxic payload DGN549-C in sodium
bisulfite. The majority of the ADC in the composition is in the
sulfonated version shown in the top panel. The bottom panel shows
an unsulfonated form of the ADC containing the anti-CD123 G4723
antibody linked to the cytotoxic payload DGN549-C (the mono-imine
structure), which can also be present in an IMGN632
composition.
[0082] FIG. 12 shows how the use of in-line process automation
technology (PAT) can be used to increase control and detectability.
Changes during steady-state operation are used to detect issues
before product quality is impacted. Ensure robust process
performance across individual unit operations with multiple PAT
modules used in combination.
TFF: FlowVPE, UV sensors, pH meter, conductivity meter, pressure
sensors, and flow meters. 1. Post-Conjugation feed: protein
concentration at expected reaction concentration with high free
drug levels. 2. Stage I: Diafiltration against reaction buffer at
pH 7.6, Free drug drops and [Protein] remains constant, pH high. 3.
Stage II: Diafiltration against formulation buffer at pH 5.0,
[Protein] remains constant, slight drop in Free Drug, and pH drops
to ensure buffer-exchange.
[0083] FIG. 13 shows a schematic of heating and cooling reactors
that could be used to pulse a conjugation reaction with an elevated
temperature. In the Pulsing Reactors (labeled "PR"), the jacket
temperature is elevated so that the reaction components contained
within the coil are heated temporarily to induce a short
temperature excursion. Then, in the Cooling Reactors (labeled
"CR"), the jacket temperature is kept at a cooler temperature to
let the reaction components within the coil cool back down from the
elevated temperature. This can reduce the amount of aggregation
occurring during the reaction. The Residence Time Reactor (RTR)
maintains the desired reaction temperature after pulsing is
complete, and its volume can be based on the desired reaction
time.
[0084] FIG. 14 shows the rate of IMGN853 conjugation (left panel)
and the accumulation of high molecular weight (HMW; aggregate)
species in conjugation reactions exposed to either a continuous
20.degree. C. temperature or to repeated pulses at higher
temperatures.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0085] The terms "antibody drug conjugate" (ADC) and
"immunoconjugate" as used herein refer to a compound or a
derivative thereof that is linked to a cell binding agent (e.g., an
antibody or antigen-binding fragment thereof) and is defined by a
generic formula: D-L-A, wherein D=cytotoxic drug, L=linker, and
A=antibody or antibody fragment. ADCs can also be defined by the
generic formula in reverse order: A-L-D. An ADC can comprise
multiple drugs and linkers per antibody or antigen-binding fragment
thereof, e.g., (D-L).sub.4-A or A-(L-D).sub.2. The terms "antibody
drug conjugate" and "immunoconjugate" are used interchangeably
herein.
[0086] A "linker" is any chemical moiety that is capable of linking
a drug to a cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) in a stable, covalent manner. Linkers can be
susceptible to or be substantially resistant to acid-induced
cleavage, light-induced cleavage, peptidase-induced cleavage,
esterase-induced cleavage, and disulfide bond cleavage, at
conditions under which the compound or the antibody remains active.
Suitable linkers are well known in the art and include, for
example, disulfide groups, thioether groups, and peptide
linkers.
[0087] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, chimeric
antibodies, humanized antibodies, human antibodies, fusion proteins
comprising an antibody, and any other modified immunoglobulin
molecule so long as the antibodies exhibit the desired biological
activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses
(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
based on the identity of their heavy-chain constant domains
referred to as alpha, delta, epsilon, gamma, and mu, respectively.
The different classes of immunoglobulins have different and well
known subunit structures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as
toxins, radioisotopes, etc.
[0088] The term "antibody fragment" refers to a portion of an
intact antibody. An "antigen-binding fragment" refers to a portion
of an intact antibody that binds to an antigen. An antigen-binding
fragment can contain the antigenic determining variable regions of
an intact antibody. Examples of antibody fragments include, but are
not limited to Fab, Fab', F(ab')2, and Fv fragments, linear
antibodies, and single chain antibodies. Antibody fragments can be
naked or conjugated to other molecules such as toxins,
radioisotopes, etc.
[0089] As used herein, the terms "variable region" or "variable
domain" are used interchangeably and are common in the art. The
variable region typically refers to a portion of an antibody,
generally, a portion of a light or heavy chain, typically about the
amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in
the mature heavy chain and about 90 to 115 amino acids in the
mature light chain, which differ extensively in sequence among
antibodies and are used in the binding and specificity of a
particular antibody for its particular antigen. The variability in
sequence is concentrated in those regions called complementarity
determining regions (CDRs) while the more highly conserved regions
in the variable domain are called framework regions (FR). Without
wishing to be bound by any particular mechanism or theory, it is
believed that the CDRs of the light and heavy chains are primarily
responsible for the interaction and specificity of the antibody
with antigen. In certain embodiments, the variable region is a
human variable region. In certain embodiments, the variable region
comprises rodent or murine CDRs and human framework regions (FRs).
In particular embodiments, the variable region is a primate (e.g.,
non-human primate) variable region. In certain embodiments, the
variable region comprises rodent or murine CDRs and primate (e.g.,
non-human primate) framework regions (FRs).
[0090] The terms "VL" and "VL domain" are used interchangeably to
refer to the light chain variable region of an antibody.
[0091] The terms "VH" and "VH domain" are used interchangeably to
refer to the heavy chain variable region of an antibody.
[0092] The term "Kabat numbering" and like terms are recognized in
the art and refer to a system of numbering amino acid residues in
the heavy and light chain variable regions of an antibody or an
antigen-binding fragment thereof. In certain aspects, CDRs can be
determined according to the Kabat numbering system (see, e.g.,
Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391 and
Kabat E A et al., (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242). Using the Kabat numbering
system, CDRs within an antibody heavy chain molecule are typically
present at amino acid positions 31 to 35, which optionally can
include one or two additional amino acids, following 35 (referred
to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid
positions 50 to 65 (CDR2), and amino acid positions 95 to 102
(CDR3). Using the Kabat numbering system, CDRs within an antibody
light chain molecule are typically present at amino acid positions
24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino
acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs
of the antibodies described herein have been determined according
to the Kabat numbering scheme.
[0093] Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end
of the Chothia CDR-H1 loop when numbered using the Kabat numbering
convention varies between H32 and H34 depending on the length of
the loop (this is because the Kabat numbering scheme places the
insertions at H35A and H35B; if neither 35A nor 35B is present, the
loop ends at 32; if only 35A is present, the loop ends at 33; if
both 35A and 35B are present, the loop ends at 34). The AbM
hypervariable regions represent a compromise between the Kabat CDRs
and Chothia structural loops, and are used by Oxford Molecular's
AbM antibody modeling software.
TABLE-US-00001 Loop Kabat AbM Chothia LI L24-L34 L24-L34 L24-L34 L2
L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 Hl H31-H35B
H26-H35B H26-H32 . . . 34 (Kabat Numbering) Hl H31-H35 H26-H35
H26-H32 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102
H95-H102 H95-H102
[0094] As used herein, the term "constant region" or "constant
domain" are interchangeable and have its meaning common in the art.
The constant region is an antibody portion, e.g., a carboxyl
terminal portion of a light and/or heavy chain which is not
directly involved in binding of an antibody to antigen but which
can exhibit various effector functions, such as interaction with
the Fc receptor. The constant region of an immunoglobulin molecule
generally has a more conserved amino acid sequence relative to an
immunoglobulin variable domain. In certain aspects, an antibody or
antigen-binding fragment comprises a constant region or portion
thereof that is sufficient for antibody-dependent cell-mediated
cytotoxicity (ADCC).
[0095] As used herein, the term "heavy chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
alpha (.alpha.), delta (.delta.), epsilon (.epsilon.), gamma
(.gamma.), and mu (.mu.), based on the amino acid sequence of the
constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM
classes of antibodies, respectively, including subclasses of IgG,
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4. Heavy chain
amino acid sequences are well known in the art. In specific
embodiments, the heavy chain is a human heavy chain.
[0096] As used herein, the term "light chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
kappa (.kappa.) or lambda (.lamda.) based on the amino acid
sequence of the constant domains. Light chain amino acid sequences
are well known in the art. In specific embodiments, the light chain
is a human light chain.
[0097] The term "conjugation process" as used herein refers to a
process in which conjugation reaction reagents (e.g., a
cell-binding agent, drug, and linker; a cell-binding agent and a
drug attached to a linker; or a cell-binding agent attached to a
linker and a drug) are mixed under conditions which allow the
reagents to react and form ADCs.
[0098] The term "batch conjugation process" as used herein refers
to a conjugation process in which the conjugation reaction reagents
are mixed together in bulk, the conjugation reaction occurs to form
conjugation reaction products (ADCs), and the conjugation reaction
products (ADCs) are then removed in bulk.
[0099] The term "continuous conjugation process" as used herein
refers to a conjugation process in which one or more conjugation
reaction reagents continue to be added to a conjugation reaction
while the conjugation reaction proceeds and after at least one
conjugation reaction product (ADC) has formed. The conjugation
reaction products (ADCs) can continue to be removed from the
conjugation reaction as the conjugation reaction proceeds.
[0100] The term "in-situ reaction" as used herein refers to a
process in which a drug and linker are mixed to form a drug
attached to a linker. The drug attached to the linker can then be
used in a conjugation reaction with an cell-binding agent (e.g., an
antibody) to form an ADC.
[0101] The term "flow reactor" as used herein refers to any reactor
vessel, typically tube like, that is used for continuous reaction
chemistry. Flow reactors can be made of stainless steel, glass,
polymers, etc.
[0102] The term "in-line monitoring" as used herein refers to
monitoring an analyte in real time, e.g., while a conjugation
reaction, concentration process, purification process, or buffer
exchange process is occurring.
[0103] The term "filter" as used herein refers to a selective
barrier that permits the separation of species in a fluid.
Separation is achieved by selectively passing (permeating) one or
more species of the fluid through the filter while retarding the
passage of one or more other species.
[0104] The term "feed stream" as used herein refers to a fluid
being fed to a filter or membrane for separation of components in
the filter or membrane.
[0105] The term "retentate" as used herein refers to the portion of
the feed stream that does not pass through the filter.
[0106] The term "permeate" as used herein refers to the portion of
the feed stream that does pass through the filter.
[0107] The term "tangential flow filtration" (TFF) as used herein
refers to a membrane-based filtration process in which a feed
stream passes parallel to a membrane face. One portion of the feed
stream passes through the membrane (permeate) while the remainder
(retentate) is recirculated back to the feed reservoir. TFF is also
referred to as cross-flow filtration. Systems for performing TFF
are known and include, for example, a Pellicon-type system
(Millipore, Billerica, Mass.), a Sartocon Cassette system
(Sartorius A G, Edgewood, N.Y.), and a Centrasette-type system
(Pall Corporation, East Hills, N.Y.).
[0108] The term "single-pass tangential flow filtration" (SPTFF) as
used herein refers to a tangential flow filtration process in which
a feedstream passes over the filtration membrane only once. Systems
for performing SPTFF are know and include, for example, a
Cadance-type system (Pall Corporation, Westborough, Mass.). Systems
and methods for performing SPTFF are disclosed, for example, in
U.S. Pat. Nos. 7,384,549, 7,510,654, 7,682,511, 7,967,987,
8,157,999, and 8,231,787, each of which is herein incorporated by
reference in its entirety.
[0109] The term "continuous diafiltration" as used herein refers to
a diafiltration process in which selective separation of solutes is
achieved in a continuous fashion by mixing a feed stream with a
diluent and pumping it across a membrane with the permeate and
retentate being removed. The product is not formed in a vessel as
filtration progresses; instead it is continuously withdrawn from
the system during the course of the filtration. "Countercurrent
diafiltration" refers to a continuous diafiltration process in
which a process stream (e.g., permeate or retentate) is recycled in
diafiltration steps.
[0110] The term "in-line monitoring" refers to monitoring an
analyte in real-time, e.g., during a production or purification
process. In-line monitoring is distinguished from in-process
sampling or offline analysis, which do not provide real-time
feedback.
[0111] The term "in-line process automation technology" refers to
any in-line measurement device used to monitor an analyte during a
process.
[0112] The term "analyte" as used herein is a broad term, and it
refers without limitation to a substance or chemical constituent in
a fluid that can be analyzed. Analytes may include naturally
occurring substances, artificial substances, metabolites, and/or
reaction products. In some embodiments, the analyte for measurement
in the methods disclosed herein is an antibody or antigen-binding
fragment thereof, drug, linker, linker bound to antibody or
antigen-binding fragment thereof, linker bound to drug,
antibody-drug conjugate (ADC), drug-to-antibody ratio (DAR), and/or
impurity.
[0113] The term "reaction buffer" as used herein refers to a buffer
in which the reaction can take place. Thus, the terms "conjugation
reaction buffer" or "conjugation buffer" as used herein refer to a
buffer in which a conjugation reaction (a continuous conjugation
reaction or a batch conjugation reaction) can take place.
Similarly, the terms "in-situ reaction buffer" or "in-situ buffer"
as used herein refer a buffer in which an in-situ reaction can take
place.
[0114] The term "formulation buffer" as used herein refers to a
buffer that permits biological activity of the active ingredient
and which contains no additional components that are unacceptably
toxic to a subject to which the formulation would be
administered.
[0115] The term "indolinobenzodiazepine" (IGN) as used herein
refers to a compound having an indolinobenzodiazepine core
structure. The indolinobenzodiazepine can be substituted or
unsubstituted. It also includes a compound having two
indolinobenzodiazepine core linked by a linker. The imine
functionality (--C.dbd.N--) as part of indolinobenzodiazepine core
can be reduced. In certain embodiments, the indolinobenzodiazepine
compound comprises a core structure represented by
##STR00001##
which can be optionally substituted.
[0116] In some embodiments, the indolinobenzodiazepine compound
comprises a core structure represented by
##STR00002##
which can be further substituted.
[0117] The term "pyrrolobenzodiazepine" (PBD) as used herein refers
to a compound having a pyrrolobenzodiazepine core structure. The
pyrrolobenzodiazepine can be substituted or unsubstituted. It also
includes a compound having two pyrrolobenzodiazepine core linked by
a linker. The imine functionality (--C.dbd.N--) as part of
indolinobenzodiazepine core can be reduced. In certain embodiments,
the pyrrolobenzodiazepine compound comprises a core structure
represented by
##STR00003##
which can be optionally substituted. In certain embodiments, the
pyrrolobenzodiazepine compounds comprises a core structure
represented by
##STR00004##
which can be optionally substituted.
[0118] As used in the present disclosure and claims, the singular
forms "a," "an," and "the" include plural forms unless the context
clearly dictates otherwise.
[0119] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0120] The term "and/or" as used in a phrase such as "A and/or B"
herein is intended to include both "A and B," "A or B," "A," and
"B." Likewise, the term "and/or" as used in a phrase such as "A, B,
and/or C" is intended to encompass each of the following
embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and
C; A and B; B and C; A (alone); B (alone); and C (alone).
[0121] H. Continuous Conjugation, Single-Pass Tangential Flow
Filtration, and Countercurrent Diafiltraion
[0122] Provided herein are continuous methods for forming and/or
processing antibody drug conjugates (ADC). In batch formation and
processing, components are added to a process, the process proceeds
for a period of time, and then the products of the process are
removed in bulk. In contrast, in the continuous methods provided
herein components continue to be added to an ongoing process, and
products can be removed throughout the process instead of in bulk
at the end of the process. For example, in a continuous conjugation
process, one or more conjugation reaction reagents continue to be
added to a conjugation reaction while the reaction proceeds and
after at least one conjugation reaction product (ADC) has formed.
Similarly, in downstream continuous concentration, purification,
and/or buffer exchange processes, ADCs, buffers, and/or other
components continue to be added to the concentration purification,
and/or buffer exchanges processes as those processes proceed, and
concentrated, purified, and buffer exchanged ADCs can be
continuously removed from those ongoing processes. Accordingly, an
entire ADC process from ADC conjugation to ADC formulation can be
continuous (see e.g., FIG. 1 (bottom)).
[0123] The present inventors have demonstrated that a conjugation
process can be performed continuously using flow reactors. Flow
reactors allow one or more conjugation reaction reagents to be
continuously added to a conjugation reaction while the conjugation
reaction proceeds and after at least one conjugation reaction
product (ADC) has formed The use of flow reactors in the
conjugation process allows for control of the conjugation reaction
and versatility of the conjugation process (e.g., rapid temperature
changes/tighter temperature control, mixing mediated by diffusion
therefore more uniform, and no constraints by vessels or suite
limitations) and improved scalability of the ADC conjugation
process (i.e., the conventional scale-up risks of batch processing
do not apply and space-time yield is optimized (e.g., performing a
pseudo scale up (increase output) by running the current process
for a longer time)).
[0124] The present inventors have further demonstrated that
continuous ADC processing can be accomplished using single-pass
tangential flow filtration (SPTFF), which can successfully separate
unconjugated drug from ADCs. Bulk (conventional) diafiltration
involves priming a tangential flow filtration (TFF) system with a
first buffer A (the buffer that a product is initially in). The
product is then added to a retentate vessel where it mixes with the
prime volume. A feed pump for the retentate vessel pumps product
from the retentate over the TFF membrane, where it is either
retained (and returns to the retentate) or discarded to waste.
Another pump (the diafiltration pump) would feed from the vessel to
buffer B contained in a separate vessel (the buffer into which the
product will be exchanged) with a feed line from the vessel into
the retentate vessel. Both pumps are started and the product in the
retentate vessel begins passing over the TFF membrane. As buffer is
removed via the waste stream, the volume in the retentate is
maintained by adding Buffer B (in equal volume) to the retentate
vessel. As a result, the product slowly is exchanged into Buffer
B.
[0125] SPTFF uses a related concept of exchanging the product
initially in Buffer A into Buffer B. However, the product only ever
makes a single-pass over the membrane so all of the appropriate
volume of Buffer B to achieve complete buffer exchange must be
achieved. To do this, SPTFF can add Buffer B over stacked stages.
The product, therefore, passes through the membranes only once:
entering in Buffer A and exiting the module in Buffer B.
[0126] Similarly, continuous ADC processing can be accomplished
using countercurrent diafiltration.
[0127] Accordingly, continuous ADC processing methods provided
herein (e.g., using SPTFF and/or countercurrent diafiltration) can
reduce processing time, improve yield, and/or improve product
consistency as compared to batch ADC processing. Continuous ADC
processing methods provided herein (e.g., using SPTFF and/or
countercurrent diafiltration) can also eliminate hold steps used in
batch conjugation processes. Continuous ADC processing methods
provided herein (e.g., using SPTFF and/or countercurrent
diafiltration) can also allow for use of smaller equipment. SPTFF
is also advantageous because antibodies that are sensitive to
oxidation, potentially caused by shear forces, may be better suited
for SPTFF.
[0128] ADC processing can involve conjugation (formation) of the
ADC, concentration of the ADC, purification of the ADC, and/or
formulation of the ADC. Although it is particularly useful for the
entire process from ADC conjugation to formulation to be continuous
(e.g., as shown in FIG. 1), it is also possible to combine
continuous processing steps with batch processing steps (e.g., as
shown in FIGS. 2 and 3). In addition, it is possible for upstream
processing steps (e.g., the preparation of an antibody, linker,
and/or drug) to be continuous and to feed continuously into the ADC
conjugation.
[0129] Accordingly, in some methods provided herein the conjugation
process for forming antibody drug conjugates (ADCs) is continuous.
In a continuous conjugation process one or more conjugation
reaction reagents continue to be added to a conjugation reaction
while the conjugation reaction proceeds and after at least one
conjugation reaction product (ADC) has formed. The conjugation
reaction reagents can be put into the system while assembled ADCs
are removed from the system. For example, in some continuous
conjugation processes provided herein, a cell binding agent (e.g.,
antibody or antigen-binding fragment thereof), a drug attached to a
linker, and a conjugation reaction buffer continue to be added to
the conjugation reaction while the conjugation reaction proceeds
and after at least one ADC is formed. In some continuous
conjugation processes provided herein, a cell binding agent (e.g.,
antibody or antigen-binding fragment thereof) attached to a linker,
a drug, and a conjugation reaction buffer continue to be added to
the conjugation reaction while the conjugation reaction proceeds
and after at least one ADC is formed. In some continuous
conjugation processes provided herein, a cell binding agent (e.g.,
antibody or antigen-binding fragment thereof), a drug, a linker,
and a conjugation reaction buffer continue to be added to the
conjugation reaction while the conjugation reaction proceeds and
after at least one ADC is formed. The reagents that continue to be
added can be added together in a single feed stream or can be fed
separately to a collection vessel or directly into a reaction
vessel.
[0130] In some continuous conjugation processes provided herein,
only one of a cell binding agent (e.g., an antibody or
antigen-binding fragment thereof), a cell binding agent attached to
a linker, a drug, a drug attached to a linker, a linker, or a
conjugation reaction buffer continues to be added to the
conjugation reaction while the conjugation reaction proceeds and
after at least one ADC is formed. In some continuous conjugation
processes provided herein, two reagents selected from the group
consisting of: a cell binding agent (e.g., an antibody or
antigen-binding fragment thereof), a cell binding agent attached to
a linker, a drug, a drug attached to a linker, a linker, and a
conjugation reaction buffer continue to be added to the conjugation
reaction while the conjugation reaction proceeds and after at least
one ADC is formed.
[0131] The use of SPTFF can allow for continuous addition and/or
removal of components from the conjugation reaction. Thus, SPTFF
can be used to prepare reagents for ADC conjugation and for
processing assembled ADCs. SPTFF can enable continuous ADC
processing so that all (or a subset) of the processing steps for a
particular ADC (e.g., production, concentration, purification,
and/or formulation) can be occur simultaneously. Countercurrent
diafilatration can also be used to prepare reagents for ADC
conjugation and for processing assembled ADCs.
[0132] According to the methods provided herein, SPTFF can be used
to concentrate an ADC, to purify an ADC, and/or to formulate an ADC
(e.g., by exchanging an ADC into a formulation buffer). SPTFF can
be used to transfer an ADC from a first buffer to a second buffer.
Countercurrent diafiltration can also be used to concentrate an
ADC, to purify an ADC, and/or to formulate an ADC (e.g., by
exchanging an ADC into a formulation buffer), and countercurrent
diafiltration can also be used to transfer an ADC from a first
buffer to a second buffer.
[0133] In some methods provided herein, SPTFF is used throughout
ADC production, purification, and formulation, making the entire
process from ADC production to formulation continuous. In some
methods provided herein, countercurrent diafilitration is used
throughout ADC production, purification, and formulation, making
the entire process from ADC production to formulation continuous.
Thus, the entire processes shown in exemplary FIGS. 4 and 10 can be
continuous.
[0134] In some methods provided herein, SPTFF is used in
combination with conventional TFF such that some portions of the
processes shown in exemplary FIGS. 4 and 10 are continuous, whereas
other portions are performed in batches. For example, an antibody
or antigen-binding fragment thereof can be buffer-exchanged prior
to conjugation using conventional (batch) TFF (see e.g., TFF1 in
FIG. 4) and then fed into a continuous process, wherein SPTFF is
used for downstream processes (see e.g., TFF2 Stage 1 and II in
FIG. 4). Countercurrent diafilitration can be used in place of or
in combination with SPTFF in such methods.
[0135] In FIGS. 4 and 10, each of the boxes shown in doted lines
represents an individual portion of the process that can be
conducted in either a batch or a continuous manner. For example,
the process for putting an antibody in buffer for conjugation shown
in the upper left hand box of FIG. 4 can use SPTFF and be performed
in a continuous fashion or can use conventional TFF and be
performed in a batch fashion. Regardless of whether the process for
putting an antibody in buffer for conjugation is performed in a
batch or continuous fashion, the ADC concentration and purification
processes shown in the box downstream of the conjugation reaction
can be use SPTFF and be performed in a continuous fashion or can
use conventional TFF and be performed in a batch fashion.
Similarly, regardless of whether the process for putting an
antibody in buffer for conjugation is performed in a batch or
continuous process, and regardless of whether the ADC is
concentrated and purified using a batch or continuous process, the
ADC can be formulated using SPTFF in a continuous fashion or using
conventional TFF in a batch fashion. Ccountercurrent diafilitration
can be used in place of or in combination with SPTFF in such
methods.
[0136] In some embodiments, at least two steps in an ADC process
are performed using SPTFF. For example, in some embodiments, SPTFF
is used to transfer an antibody or antigen-binding fragment thereof
into a conjugation buffer and used to concentrate and purify the
ADC after it is formed, while either SPTFF or TFF is used to
exchange the ADC into formulation buffer. In some embodiments,
SPTFF is used to transfer an antibody or antigen-binding fragment
thereof into a conjugation buffer and used to exchange a purified
ADC into formulation buffer, while either SPTFF or TFF is used to
concentrate and purify the ADC after it is formed. In some
embodiments, SPTFF is used to concentrate and purify the ADC and
used to exchange the concentrated and purified ADC into formulation
buffer, wherein either SPTFF or TFF is used to transfer the
antibody or antigen-binding fragment thereof into a conjugation
buffer.
[0137] SPTFF can use an ultrafiltration membrane, e.g., in methods
of concentrating an ADC. SPTFF can use a diafiltration membrane,
e.g., in methods of purifying an ADC and/or in methods of
transferring an ADC to a buffer (e.g., a formulation buffer).
[0138] In some embodiments, at least two steps in an ADC process
are performed using SPTFF and/or countercurrent diafilitration. For
example, in some embodiments, SPTFF and/or countercurrent
diafilitration is used to transfer an antibody or antigen-binding
fragment thereof into a conjugation buffer and used to concentrate
and purify the ADC after it is formed, while SPTFF, countercurrent
diafilitration, and/or TFF is used to exchange the ADC into
formulation buffer. In some embodiments, SPTFF and/or
countercurrent diafilitration is used to transfer an antibody or
antigen-binding fragment thereof into a conjugation buffer and used
to exchange a purified ADC into formulation buffer, while SPTFF,
countercurrent diafilitration, and/or TFF is used to concentrate
and purify the ADC after it is formed. In some embodiments, SPTFF
and/or countercurrent diafilitration is used to concentrate and
purify the ADC and used to exchange the concentrated and purified
ADC into formulation buffer, wherein SPTFF, countercurrent
diafilitration, and/or TFF is used to transfer the antibody or
antigen-binding fragment thereof into a conjugation buffer.
[0139] Column chromatography can also be used in a flow-through
mode in the continuous ADC processing methods provided herein
(e.g., in combination with SPTFF and/or countercurrent
diafilitration). For example, a conjugation reaction (e.g., a
continuous conjugation reaction) can feed into flow-through column
chromatography to remove unconjugated drug from a conjugation
reaction (similar to the role of the TFF2, Stage ILDF step in FIG.
4). The ADCs purified via the flow-through column chromatography
can then feed into an SPTFF process for buffer exchange into a
formulation buffer (e.g., the TFF2, Stage II ILDF step in FIG. 5.)
The ADCs purified via the flow-through column chromatography can
also feed into a countercurrent diafiltration process for buffer
exchange into a formulation buffer.
[0140] In some instances, the reaction parameters of the continuous
flow conjugation processes provided herein can be rapidly changed
or "pulsed". For example, in a continuous flow conjugation,
temperature can be rapidly altered, e.g., by using a water bath,
encapsulated reactor, heater, thermoelectric source, and/or
insulating a section of coils and/or tubes through which the
reaction flows. In addition, in a continuous flow conjugation, pH
can be rapidly altered, e.g., by addition of an acid or base.
Accordingly, in certain instances, a conjugation reaction is
performed using a pulsed parameter. The use of a pulsed parameter
can, for example, decrease reaction time (i.e., increase reaction
speed), without compromising product quality, quench or stop a
reaction temporarily by rapidly dropping the temperature, stabilize
the conjugate in solution before another perturbation (e.g.,
addition of another chemical reagent) is introduced, whereas longer
exposures to the same parameter can dramatically decrease product
quality or product stability.
[0141] In certain instances, the conjugation reaction is exposed to
an altered temperature (e.g., increased or decreased) for a
specified time increment for a specified number of times. For
example, in one instance, temperature is increased by at least
2.degree. C., at least 3.degree. C., at least 4.degree. C., or at
least 5.degree. C. Accordingly, the temperature can be increased or
decreased by at least 5.degree. C., 10.degree. C., 15.degree. C.,
20.degree. C., 25.degree. C., 30.degree. C., or 35.degree. C. For
instance, the temperature can be increased or decreased by
5.degree. C., 10.degree. C., 15.degree. C., 20.degree. C.,
25.degree. C., 30.degree. C., or 35.degree. C. The temperature can
also be increased or decreased by about 5.degree. C. to about
10.degree. C., by about 10.degree. C. to about 15.degree. C., by
about 15.degree. C. to about 20.degree. C., by about 20.degree. C.
to about 25.degree. C., by about 25.degree. C. to about 30.degree.
C., or by about 30.degree. C. to about 35.degree. C. Thus, for
example, temperature can be increased (e.g., from about 20.degree.
C.) to an elevated temperature of 25.degree. C., 30.degree. C.,
35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., or
55.degree. C. Temperature can be also increased (e.g., from about
25.degree. C.) to an elevated temperature of 30.degree. C.,
35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., or
55.degree. C. In certain instances, the temperature does not exceed
55.degree. C. In certain instance, the temperature is increased
(e.g., from about 20.degree. C.) to an elevated temperature in the
range of about 35.degree. C. to about 55.degree. C. or to an
elevated temperature in the range of about 40.degree. C. to about
50.degree. C. In certain instances, the temperature is increased
(e.g., from about 20.degree. C.) to an elevated temperature of
about 60.degree. C., to about 70.degree. C., to about 80.degree.
C., to about 90.degree. C., or to about 100.degree. C. (e.g., for a
short time increment such as 10 seconds). In certain instances, the
temperature is increased (e.g., from about 20.degree. C.) to an
elevated temperature in the range of 60.degree. C. to 70.degree.
C., in the range of 70.degree. C. to 80.degree. C., in the range of
80.degree. C. to 90.degree. C., or in the range of 90.degree. C. to
100.degree. C. (e.g., for a short time increment such as 10
seconds). In certain instances, the time it takes to increase or
decrease the temperature to the elevated or reduced temperature is
no more than 2 minutes. In certain instances, the time it takes to
increase or decrease the temperature to the elevated or reduced
temperature is no more than 1 minute.
[0142] In certain instances, the conjugation reaction is exposed to
an altered pH (e.g., increased or decreased) for a specified time
increment for a specified number of times. For example, in one
instance, pH is increased or decreased by about 1, about 2, about
3, about 4, or about 5. In one instance, pH is increased by about 1
to about 2, by about 2 to about 3, by about 3 to about 4, or by
about 4 to about 5. Thus, for example, pH can be increased (e.g.,
from about 4) to about 5, about 6, about 7, about 8, or about 9. PH
can also be increased (e.g., from about 5) to about 6, about 7,
about 8, or about 9. PH can also be decreased (e.g., from about 9)
to about 8, about 7, about 6, about 5, or about 4.
[0143] In certain instances, the pulse (e.g., exposure to altered
temperature and/or pH) occurs for about 30 seconds, about 1 minute,
about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes,
about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes,
about 10 minutes, about 15 minutes, about 30 minutes, about an
hour, about 1.5 hours, or about 2 hours. The pulse (e.g., exposure
to altered temperature and/or) can also occur, for example, for
about 30 seconds to about 1 minute, for about 1 minute to about 2
minutes, for about 2 minutes to about 3 minutes, for about 3
minutes to about 4 minutes, for about 4 minutes to about 5 minutes,
for about 6 minutes to about 7 minutes, for about 7 minutes to
about 8 minutes, for about 8 minutes to about 9 minutes, or for
about 9 minutes to about 10 minutes. The pulse (e.g., exposure to
altered temperature and/or) can also occur, for example, for about
1 to 10 minutes, for about 1 to 15 minutes, for about 1 to 30
minutes, for about 1 minute to 1 hour, for about 1 minute to about
1.5 hours, or for about 1 minute to about 2 hours. The pulse (e.g.,
exposure to altered temperature and/or) can also occur, for
example, for about 1 to 5 minutes, or about 5 to 10 minutes, about
10 to about 15 minutes, about 15 minutes to about 30 minutes, about
30 minutes to about 1 hour, about 1 hour to about 1.5 hours, or
about 1.5 hours to about 2 hours. In certain instances, the pulse
(e.g., exposure to altered temperature and/or pH) does not exceed 2
hours, 1 hour, 30 minutes, 20 minutes, or 15 minutes.
[0144] In certain instances, the pulse (e.g., exposure to altered
temperature and/or pH) occurs once. In certain instances, the pulse
(e.g., exposure to altered temperature and/or) is repeated twice,
three times, four times, five times, six times, seven times, eight
times, nine times, or ten times. In certain instances, the pulse
(e.g., exposure to altered temperature and/or) occurs one to five
times. In certain instances, the pulse (e.g., exposure to altered
temperature and/or) occurs two to twenty times or five to ten
times.
[0145] Continuous ADC processing methods (e.g., using SPTFF and/or
countercurrent diafiltration) can be used with or without in-line
monitoring processes (discussed below).
III. In-Line Process Automation Technology (In-Line PAT)
[0146] Provided herein are in-line process automation technologies
used for forming and processing antibody drug conjugates (ADCs).
Such technologies provide for direct measurements, which can
eliminate off-line assays and decrease handling of materials by
operators. In-line monitoring can be used to monitor ADC (protein)
concentration as well as the removal of free drug. This can allow
targeting a final ADC concentration based on data obtained from
in-line readings instead of based on a specific volume or number of
diavolumes used in the processes (e.g., purification processes).
PAT implementation allows for increase control and detectability
(e.g., changes during steady-state operation can be used to detect
issues before product quality is impacted) and use of multiple PAT
modules (e.g., FlowVPE, UV sensors, pH meter, conductivity meter,
pressure sensors, or flow meters) can ensure robust process
performance across individual unit operations.
[0147] In-line monitoring can be used, for example, to monitor the
flow rates in a feed stream from any pump in, for example, an
in-situ reaction and/or an ADC conjugation reaction (e.g., a
continuous conjugation reaction or a batch conjugation reaction).
In-line monitoring can be used, for example, to monitor the
concentration of a component added to an in-situ reaction and/or an
ADC conjugation reaction (e.g., a continuous conjugation reaction
or a batch conjugation reaction). Such monitoring can ensure
adequate control over the stoichiometry of the reactions. The
in-line monitoring can monitor the flow rate or concentration of,
for example, an antibody or antigen-binding fragment thereof, a
drug, a linker, drug attached to a linker, an antibody or
antigen-binding fragment thereof attached to a linker, and/or a
conjugation buffer. The in-line monitoring can monitor the flow
rate of concentration of an antibody or antigen-binding fragment
thereof into a conjugation reaction buffer.
[0148] In-line monitoring can also be used, for example, to
determine when to stop a conjugation reaction, e.g., by stopping to
add conjugation buffer, by stopping the circulation of conjugation
buffer, and/or starting to rinse or remove conjugation buffer. In
some embodiments provided herein, in-line monitoring of an
unconjugated drug or an unconjugated drug attached to a linker can
be used. Measurements of unconjugated drug or unconjugated drug
attached to a linker can be used to infer the average number of
drugs per antibody (DAR) achieved in a conjugation reaction. Thus,
a conjugation reaction can be stopped when the targeted DAR is
reached.
[0149] In-line monitoring can also be used to monitor the
concentration and/or purification of an ADC. For example, in-line
monitoring can be used before or after the ILC and/or TFF2 ILDF
processes shown in exemplary FIG. 4 or before or after the ILC or
TFF3 ILDF processes shown in exemplary FIG. 10. The concentration
and/or purification can use filtration (e.g., ultrafiltration,
difiltration). The filtration can be tangential flow filtration,
including single-pass tangential flow filtration (SPTFF). The
filtration can be countercurrent diafiltration. When used to
monitor filtration, in-line monitoring can be used to measure an
analyte in either the retentate or the permeate. Thus, for example,
in-line monitoring of an unconjugated drug or unconjugated drug
attached to linker in a retentate can be used to assess the degree
of purification of the ADC. Levels of unconjugated drug or
unconjugated drug attached to a linker can be high in a retentate
shortly after a conjugation reaction but low in a retentate after
purification (see e.g., FIG. 12). In-line monitoring of an ADC in a
retentate or a permeate can be used to assess ADC loss during
concentration and/or purification processes (see e.g., FIG.
12).
[0150] The purification can also use chromatography (e.g., flow
through column chromatography). In-line monitoring at the end of a
chromatography column can, for example, measure ADC levels and can
be used to determine when a column is overloaded or when there is
ADC breakthrough.
[0151] In-line monitoring can also be used to measure pH, which can
be used, for example, to determine the completeness of a buffer
exchange (see e.g., FIG. 12).
[0152] Exemplary in-line monitoring technologies include the use
of, for example, a Fourier Transform Infared (FTIR) flow cell, High
Performance Liquid Chromatography (HPLC), or Ultra Performance
Liquid Chromatography (UPLC).
[0153] In-line monitoring technologies can use, for example, a
FlowVPE or UV sensor. In some embodiments, FlowVPE is used to
perform in-line monitoring. FlowVPE uses a flow cell for continuous
monitoring.
[0154] The efficacy of tangential flow filtration (e.g.,
single-pass tangential flow filtration (SPTFF) can be monitored
in-line using FlowVPE, a UV sensor, a pH meter, a conductivity
meter, a pressure sensor, a flow meter, etc. The efficacy of
countercurrent diafiltration can also be monitored in-line using
FlowVPE, a UV sensor, a pH meter, a conductivity meter, a pressure
sensor, a flow meter, etc.
[0155] In-line monitoring processes can be used in combination with
continuous conjugation processes (discussed above), e.g., using
single-pass tangential flow filtration and/or countercurrent
diafiltration, or with batch conjugation processes (which are known
in the art).
IV. Antibody Drug Conjugates (ADCs)
[0156] As provided herein, antibody drug conjugates (ADCs) can
comprise a cell-binding agent, a linker, and a drug.
[0157] The cell-binding agent can be an antibody or antigen-binding
fragment thereof, e.g., a monoclonal antibody or antigen-binding
fragment thereof. The cell-binding agent (e.g., antibody or
antigen-binding fragment thereof) can be humanized. The
cell-binding agent (e.g., antibody or antigen-binding fragment
thereof) can be human.
[0158] The cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) can specifically bind to human CD37, CD33, FOLR1,
CD123, CD19, cMET, ADAM9, or HER2.
[0159] The cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) can comprise the six CDRs of an antibody provided
in Table 1.
TABLE-US-00002 TABLE 1 Antibody Complementarity Determining Region
Sequences Name CDR Sequence huMov19 VH CDR1 GYFMN (SEQ ID NO: 1)
huMov19 VH CDR2 RIHPYDGDTFYNQKFQG (SEQ ID NO: 2) huMov19 VH CDR3
YDGSRAMDY (SEQ ID NO: 3) huMov19 VL CDR1 KASQSVSFAGTSLMH (SEQ ID
NO: 4) huMov19 VL CDR2 RASNLEA (SEQ ID NO: 5) huMov19 VL CDR3
QQSREYPYT (SEQ ID NO: 6) Z4681A VH CDR1 SYYIH (SEQ ID NO: 7) Z4681A
VH CDR2 VIYPGNDDISYNQKFQG (SEQ ID NO: 8) Z4681A VH CDR3 EVRLRYFDV
(SEQ ID NO: 9) Z4681A VL CDR1 KSSQSVFFSSSQKNYLA (SEQ ID NO: 10)
Z4681A VL CDR2 WASTRES (SEQ ID NO: 11) Z4681A VL CDR3 HQYLSSRT (SEQ
ID NO: 12) G4723A VH CDR1 SSIMH (SEQ ID NO: 13) G4723A VH CDR2
YIKPYNDGTKYNEKFKG (SEQ ID NO: 14) G4723A VH CDR3 EGGNDYYDTMDY (SEQ
ID NO: 15) G4723A VL CDR1 RASQDINSYLS (SEQ ID NO: 16) G4723A VL
CDR2 RVNRLVD (SEQ ID NO: 17) G4723A VL CDR3 LQYDAFPYT (SEQ ID NO:
18) huCMET-27 VH CDR1 SYDMS (SEQ ID NO: 19) huCMET-27 VH CDR2
TINSNGVSIYYPDSVKG (SEQ ID NO: 20) huCMET-27 VH CDR3 EEITTEMDY (SEQ
ID NO: 21) huCMET-27 VL CDR1 RASESVDSYGNSFIH (SEQ ID NO: 22)
huCMET-27 VL CDR2 RASNLES (SEQ ID NO: 23) huCMET-27 VL CDR3
QQSNEEPLT (SEQ ID NO: 24) huB4 VH CDR1 SNWMH (SEQ ID NO: 25) huB4
VH CDR2 EIDPSDSYTN (SEQ ID NO: 26) huB4 VH CDR3 GSNPYYYAMDY (SEQ ID
NO: 27) huB4 VL CDR1 SASSGVNYMH (SEQ ID NO: 28) huB4 VL CDR2
DTSKLAS (SEQ ID NO: 29) huB4 VL CDR3 HQRGSYT (SEQ ID NO: 30)
huADAM9 VH CDR1 SYWMH (SEQ ID NO: 31) huADAM9 VH CDR2
EIIPIFGHTNYNEKFKS (SEQ ID NO: 32) huADAM9 VH CDR3 GGYYYYFNSGTLDY
(SEQ ID NO: 33) huADAM9 VL CDR1 KASQSVDYSGDSYMN (SEQ ID NO: 34)
huADAM9 VL CDR2 AASDLES (SEQ ID NO: 35) huADAM9 VL CDR3 QQSHEDPFT
(SEQ ID NO: 36)
[0160] The cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) can comprise the variable heavy chain and/or
variable light chain of an antibody provided in Table 2. In some
embodiments, the cell-binding agent (e.g., antibody or
antigen-binding fragment thereof) comprises the CDRs of (e.g., the
Kabat-defined, AbM-defined, or Chothia-defined CDRs) a variable
heavy and variable light chain of an antibody provided in Table
2.
TABLE-US-00003 TABLE 2 Antibody Variable Heavy and Variable Light
Sequences Name Variable Heavy or Variable Light Sequence huMov19 VH
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMN
WVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATL
TVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAM DYWGQGTTVTVSS (SEQ ID NO: 37)
huMov19 VL version 1.00 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLM
HWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSK
TDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKL EIKR (SEQ ID NO: 38) huMov19
VL version 1.60 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLM
HWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSK
TDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKL EIKR (SEQ ID NO: 39) Z4681A VH
QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIH
WIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATL
TADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYF DVWGQGTTVTVSS (SEQ ID NO: 40)
Z4681A VL EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKN
YLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGS
GTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLE IKR (SEQ ID NO: 41) G4723A VH
QVQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMH
WVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRAT
LTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDY YDTMDYWGQGTLVTVSS (SEQ ID NO:
42) G4723A VL DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWF
QQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDY
TLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIK R (SEQ ID NO: 43) huCMET-27 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMS
WVRQAPGKGLEWVATINSNGVSIYYPDSVKGRFTI
SRDNAKNSLYLQMNSLRAEDTAVYYCAREEITTEM DYWGQGTLVTVSS (SEQ ID NO: 44)
huCMET-27 VL EIVLTQSPATLSLSPGERATLSCRASESVDSYGNSFI
HWYQQKPGQAPRLLIYRASNLESGIPARFSGSGSGT
DFTLTISSLEPEDFAVYYCQQSNEEPLTFGQGTKVEL KR (SEQ ID NO: 45) huB4 VH
QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWM
HWVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKA
KLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPY YYAMDYWGQGTSVTVSS (SEQ ID NO:
46) huB4 VL EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWY
QQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTDYS
LTISSMEPEDAATYYCHQRGSYTFGGGTKLEIKR (SEQ ID NO: 47) huADAM9 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWMH
WVRQAPGKGLEWVGEIIPIFGHTNYNEKFKSRFTIS
LDNSKNTLYLQMGSLRAEDTAVYYCARGGYYYYF NSGTLDYWGQGTTVTVSS (SEQ ID NO:
48) huADAM9 VL DIVMTQSPDSLAVSLGERATISCKASQSVDYSGDSY
MNWYQQKPGQPPKLLIYAASDLESGIPARFSGSGSG
TDFTLTISSLEPEDFATYYCQQSHEDPFTFGQGTKLE IK (SEQ ID NO: 49)
[0161] The cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) can corn rise the heavy chain and/or light chain
of an antibody rovided in Table 3.
TABLE-US-00004 TABLE 3 Antibody Heavy and Light Sequences Name
Heavy or Light Sequence huMov19 Heavy
QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMN
WVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATL
TVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAM
DYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ
ID NO: 50) huMov19 Light 1.00 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLM
HWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSK
TDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKL
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 51) huMov19
Light 1.60 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLM
HWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSK
TDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKL
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 52) Z4681A
Heavy QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIH
WIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATL
TADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYF
DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ
ID NO: 53) Z4681A Light EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKN
YLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGS
GTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 54) G4723A Heavy
QVQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMH
WVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRAT
LTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDY
YDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLCLSPG
(SEQ ID NO: 55) G4723A Light DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWF
QQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDY
TLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 56)
[0162] In certain embodiments, an anti-FOLR1 antibody is encoded by
the plasmids deposited with the American Type Culture Collection
(ATCC), located at 10801 University Boulevard, Manassas, Va. 20110
on Apr. 7, 2010 under the terms of the Budapest Treaty and having
ATCC deposit nos. PTA-10772 and PTA-10773 or 10774.
[0163] The cell-binding agent (e.g., antibody or antigen-binding
fragment thereof) can be CD37-3, huMov19, Z4681A, G4732A, huB4,
huCMET-27, huADAM9, or Herceptin (trastuzumab).
[0164] The drug can be a cytotoxic agent. The cytotoxic agent can
be any compound that results in the death of a cell, or induces
cell death, or in some manner decreases cell viability, and
includes, for example, maytansinoids, maytansinoid analogs,
benzodiazepines (e.g. an indolino-benzodiazepine (IGN) or a
pyrrolobenzodiazepine (PBD)), taxoids, CC-1065 and CC-1065 analogs,
duocarmycins and duocarmycin analogs, enediynes, such as
calicheamicins, dolastatin and dolastatin analogs including
auristatins, tomaymycin derivaties, leptomycin derivaties,
methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin,
vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and
morpholino doxorubicin. In one embodiment, the maytansinoid can be
DM1. In another embodiment, the maytansinoid can be DM4. In one
embodiment, the indolino-benzodiazepine can be DGN462. In another
embodiment, the indolino-benzodiazepine can be DGN549. In other
embodiments, the pyrrolobenzodiazepine can be talirine, tesirine,
SJG136, or SGD1882.
[0165] Suitable linking groups are well known in the art and
include, for example, disulfide groups, thioether groups, acid
labile groups, photolabile groups, peptidase labile groups and
esterase labile groups. Linker molecules include, for example,
N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g.,
Carlsson et al., Biochem. J., 173: 723-737 (1978)), N-succinimidyl
4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.
4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate
(sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl
4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry
number 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride.
The linker can be, SMCC, sSPDB, or a peptide linker.
[0166] An ADC can contain multiple drugs per antibody. The number
of drugs per antibody is often referred to as the drug antibody
ratio (DAR). In one aspect, the number of drug molecules that can
be attached to a cell binding agent can average from about 2 to
about 8. Thus, by way of example, the processes provided herein can
target a DAR of about 3 to about 4.
[0167] In some embodiments, the ADC is "IMGN853." As used herein
"IMGN853" refers to an ADC containing the huMov19 antibody, the
sulfoSPDB linker, and the DM4 maytansinoid. HuMov19 (M9346A)
contains a heavy chain comprising the same amino acid sequence as
the amino acid sequence of the heavy chain encoded by the plasmid
deposited with the American Type Culture Collection (ATCC) as
PTA-10772 and (ii) a light chain comprising the same amino acid
sequence as the amino acid sequence of the light chain encoded by
the plasmid deposited with the ATCC as PTA-10774. IMGN853 is
described in WO2011/106528, which is herein incorporated by
reference in its entirety.
[0168] In some embodiments, the ADC is "IMGN779." As used herein,
"IMGN779" refers to an ADC containing the Z4681A antibody, the
sulfoSPDB linker, and the indolino-benzodiazepine DGN462.
[0169] In some embodiments, the ADC is "IMGN632." As used herein,
"IMGN632" refers to the ADC composition shown in FIG. 11. The ADC
composition comprises ADCs comprising an average of 1.5 to 2.1
DGN549-C cytotoxic agents per huCD123-6Gv4.7 ("G4723A") antibody in
a sulfonated version (FIG. 11, top panel). The ADC composition can
also comprise the unsulfonated ADC (the mono-imine structure shown
in FIG. 11, bottom panel).
[0170] Exemplary ADCs that bind to ADAM9 are disclosed in
PCT/US2017/067823 (published as WO2018119196), which is herein
incorporated by reference in its entirety. Exemplary ADCs that bind
to ADAM9 are also disclosed in U.S. Application No. 62/691,342,
filed Jun. 28, 2018, which is herein incorporated by reference in
its entirety. Exemplary ADCs that bind to cMET are disclosed in
PCT/US2018/012168 (published as WO2018129029), which is herein
incorporated by reference in its entirety. Exemplary ADCS that bind
to CD19 are disclosed in WO2012156455, which is herein incorporated
by reference in its entirety.
V. EXAMPLES
Example 1: A Continuous Conjugation Process for the Conjugation of
IMGN853
[0171] To reduce processing time and improve yields for large-scale
manufacturing of antibody drug conjugates (ADCs), a continuous
conjugation process was developed. In continuous conjugation, the
processing steps happen simultaneously. Recirculation of the
conjugate during purification and buffer exchange steps, as well as
intermediate hold steps that were used during batch conjugation
were eliminated using continuous conjugation. In this manner, the
process ran continuously for 4-5 days, and the conjugate was
formulated directly after passing out of the TFF purification
steps. In addition, in-line process analytical technology (PAT)
systems were used to allow for direct measurements of the
conjugate, so off-line assays and handling of material by operators
was eliminated. Further, because processing time was reduced, the
requirement for large equipment to produce large quantities of
conjugates was not necessary: smaller equipment and shorter
processing times were sufficient to produce large amounts of
product. In addition, for a given quantity of final conjugate
produced, the product quality profile was more consistent when
generated using continuous processing. By contrast, the requirement
for multiple batches in bulk conjugation introduces the potential
for increased batch-to-batch variability in product quality.
[0172] The continuous conjugation process for the conjugation of
the ADC "IMGN853" is detailed in the flow diagram represented in
FIG. 4. IMGN853 contains an anti-FOLR1 antibody ("huMov19")
conjugated to DM4 maytansinoid drugs through sulfo-SPDB linkers.
IMGN853 is described in WO 2011/106528, which is herein
incorporated by reference in its entirety.
Example 1A: Flow Chemistry Studies
[0173] Conventional IMGN853 conjugation involves an in-situ
reaction between the maytansinoid drug DM4 and the N-succinimidyl
4-(2-pyridyldithio)-2-sulfobutanoate (sSPDB) linker in 70%
N,N-dimethylacetamide (DMA). Appropriate volumes of DM4 and sSPDB
stock solution are added to the in-situ reaction vessel. Additional
DMA (solvent) is added to the in-situ vessel to target 70% DMA by
volume. The final reactant added to the in-situ reaction vessel is
reaction buffer. After the reaction buffer is added, the in-situ
reaction initiates and mixes for 60-120 minutes. The in-situ
components are then added to a conjugation vessel containing the
M9346A (huMov19) antibody that has been diafiltered into reaction
buffer. All of the components are mixed, and the conjugation
reaction proceeds with gentle mixing overnight to generate the
desired conjugate with a target drug-antibody ratio (DAR) of
3.4.
[0174] In this study, a continuous conjugation of IMGN853 was
performed. The continuous conjugation reaction used all of the same
stoichiometric IMGN853 parameters with one notable exception. The
pH of the reaction buffer for the conjugation reaction was
increased from 7.6 to 8.7. The pH of the in-situ reaction buffer
was not changed and remained at 7.6. The increase in pH was
instituted to increase the conjugation reaction kinetics. Instead
of targeting 18 hours for the conjugation reaction, the higher pH
buffer generated the conjugate in approximately 4 hours to allow
for a faster readout and more flexible experimental protocols.
[0175] For the continuous conjugation experiments, syringe pumps
were used to continuously add reagents. The set up shown in FIG. 5.
Three syringe pumps were used. The first syringe pump controlled
the addition of the in-situ components dissolved in DMA. These
included the DM4 (payload), sSPDB (linker), and DMA (additional
solvent). These three components were combined in stoichiometric
ratios and then drawn into a 2.5 mL Hamilton Gastight syringe. The
second syringe pump metered the addition of in-situ reaction
buffer. A 5 mL plastic BD syringe was used. 1/16'' PEEK tubing was
used for the continuous conjugation studies. The in-situ reaction
buffer and DMA component feed lines were fed into an in-line static
mixer to ensure adequate mixing of the two reagents. These two feed
streams combined and exited through a single piece of tubing from
the static mixer of the same tubing material. The flow rates for
the in-situ buffer and DMA components were 952.9 nL/min and 2.2233
.mu.L/min, respectively. Targeting a 90 minute in-situ reaction
required 18.6 centimeters of PEEK tubing based on the flow rate for
the two inlet streams and the cross-sectional area of the tubing
used. This length of PEEK tubing where the in-situ reaction
occurred was fed into a second in-line static mixer. The other
inlet for the second static mixer was from the third syringe pump.
The third syringe controlled the addition of the antibody and
conjugation reaction buffer (pH 8.7). The antibody and conjugation
reaction buffer were combined and drawn into a 30 mL plastic BD
syringe. The same PEEK tubing was used to feed the antibody and
conjugation reaction buffer at a flow rate of 24.615 .mu.L/min.
Similar to the in-situ static mixer, the second static mixer
ensured the conjugation reaction components were well mixed and
combined into a single exiting stream. The conjugation reactants
left the static mixer in a piece of the same PEEK tubing that was
4.3 meters long to achieve the target conjugation reaction duration
of 4 hours.
[0176] The studies were performed by filling all three of the
syringes prior to the start of any flow. First, the in-situ
reaction buffer (syringe 2) and DMA components (syringe 1) pumps
were started at the same time. After approximately 90 minutes, pump
three (antibody and conjugation reaction buffer) was started.
Approximately five hours from starting the first two pumps,
conjugate eluted from the 4.3 meter long piece of PEEK tubing.
Sample analysis was performed by collecting directly from the end
(i.e. pooled samples were collected from the well-mixed conjugate
pool). To measure the flow rates of the in-situ and conjugation
feed steams (from any pump), flow meters can further be used. Flow
meters serve as a process analytical technology (PAT) mechanism to
ensure adequate control over the stoichiometry of the
reactions.
[0177] The results of a first IMGN853 continuous conjugations study
are shown in Table 4.
TABLE-US-00005 TABLE 4 Results of a first IMGN853 continuous
conjugation study. Free Drug SEC Total Sample DAR HMW Monomer LMW
DM4-S-TBA DM4 DM4-S-Py FM Control (Batch) PC-002 3.52 1.04% 98.93%
0.04% 17.35% 4.09% 2.38% 24.72% In-Line (Early), 3.23 1.19% 98.71%
0.11% unfiltered In-Line (End), unfiltered 3.56 1.13% 98.82% 0.06%
19.33% 5.49% 2.89% 29.54% Continuous Pooled 3.58 1.20% 98.75% 0.06%
18.17% 4.52% 1.95% 25.92% (PC-002)
[0178] The study showed that the continuous conjugation product
quality was comparable to control (batch) product quality performed
side-by-side. The product quality took some time to reach
steady-state as evidenced by the lower drug-antibody ratio (DAR) at
the "early" time point. This was consistent in a second study where
the 30 minute sample also had a slightly lower DAR. By 3 hours in
the second study, the product quality had achieved steady-state at
the target DAR (as compared to the side-by-side batch control). In
addition to DAR, the product quality was also comparable in terms
of monomer and free drug levels. Together, these results indicate
the continuous conjugation process performed well and was
comparable to side-by-side batch control in terms of product
quality.
[0179] The results of a second study are shown in Table 5.
TABLE-US-00006 TABLE 5 Results of a second IMGN853 continuous
conjugation study. Free Drug SEC Total Sample DAR HMW Monomer LMW
DM4-S-TBA DM4 DM4-S-Py FM NG006_Control PC002 3.71 1.05 98.95 0.00
18.81% 4.82% 2.55% 27.06% NG006_In-line 3.21 1.09 98.91 0.00 15.56%
3.53% 0.74% 21.07% (30 min) NG006_In-line (3h) 3.68 0.95 99.05 0.00
18.78% 5.13% 3.18% 28.54% NG006_In-Line (16 h) 3.68 0.89 99.11 0.00
18.95% 5.77% 2.66% 29.02% NG006_In-Line (24 h) 3.73 0.93 99.07 0.00
18.72% 4.84% 2.81% 27.01% NG006_In-Line (40 h) 3.70 1.13 98.87 0.00
18.37% 4.43% 2.69% 26.91% NG006_Pooled, filtered 3.83 1.25 98.75
0.00 21.64% 4.03% 2.92% 29.38%
[0180] The same parameters were used in this study as the study
above, but the process was run continuously for 40 hours to show
that 100 mg equipment setup could be used to generate 10.times.
more product by running the process longer. The results showed that
between 30 minutes and 3 hours of eluting conjugate, the conjugate
reached steady-state and was comparable to the side-by-side batch
control by SEC and free drug analysis. The product quality at three
hours was nearly identical to the conjugate eluting at 40 hours
indicating the continuous process performed consistently as long as
the feed reagents were supplied to the reaction tubing.
[0181] In a manufacturing setting, pumps for each individual
reagent can be used to keep stock solutions separate. Instead of
combining DM4, sSPDB, or DMA into a single feed stream, each of the
stock solutions can be fed separately to a collection vessel or
directly into the in-situ reaction. The same can be applied for the
antibody and conjugation reaction buffer. The stock solution of
antibody can be fed using a designated pump while a separate pump
controls the addition of conjugation reaction buffer at the
appropriate volumetric ratio to the other conjugation reaction
components.
[0182] Other PAT mechanisms can be used to monitor either the
in-situ or conjugation reaction as it proceeds. A Fourier Transform
Infared (FTIR) flow cell can be used in-line to monitor the in-situ
or conjugation reaction performance. Additionally, multiple FTIR
flow cells or a similar PAT tool can be placed at intermediate
locations along the in-situ and conjugation reaction tubing to
monitor the reaction as it occurs. The formation of the desired
conjugate can be measured from the start of the reaction all the
way to the end. Any significant change in the PAT signal can then
be used to identify where in either the in-situ or conjugation
reaction (beginning, middle, or end) an error has occurred. For the
conjugate, an alternative PAT mechanism can be a rapid High
Performance Liquid Chromatography (HPLC)-based instrument (Ultra
Performance Liquid Chromatography (UPLC)) with or without a UV
sensor. The HPLC-based analysis can be used to measure the DAR
while UV sensors placed in-line can monitor the antibody
concentration in the conjugation reaction.
Example 1B: Single-Pass Tangential Flow Filtration Studies
[0183] Conventional tangential flow filtration (TFF) is used twice
during the IMGN853 drug substance process. First, TFF is performed
to buffer-exchange the formulated antibody into conjugation
reaction buffer, prior to the conjugation reaction (TFF1). After
the conjugation reaction, TFF is used to remove residual impurities
from the crude-reaction mixer and buffer-exchange the conjugate
from conjugation reaction buffer into basal formulation buffer
(TFF2). A continuous process was developed to replace both of these
conventional TFF steps with single-pass TFF (SPTFF) method.
Continuous SPTFF utilized the in-line concentration (ILC) and
in-line diafiltration (ILDF) technologies from Pall Life
Sciences.
[0184] For TFF1, the antibody was diafiltered for 8 diavolumes at
30 mg/mL during conventional processing. The incoming antibody was
formulated at 60 mg/mL and was diluted prior to diafiltering during
conventional TFF1 processing. During continuous processing, the
formulated antibody at 60 mg/mL was fed into a T-connector, where
another pump added conjugation reaction buffer to dilute the
antibody to the target concentration of 30 mg/mL. After the
T-connector, a single-feed stream of 30 mg/mL antibody was fed into
the ILDF module. The ILDF diafiltration buffer pump controlled the
addition of conjugation reaction buffer to the ILDF module so that
the buffer-exchange was achieved in a single pass. The antibody
exiting the ILDF was at 30 mg/mL in the conjugation reaction
buffer.
[0185] PAT control can be implemented at various places to monitor
and control this unit operation. A FlowVPE or UV sensor can be
placed on the formulated antibody feed stream (60 mg/mL), diluted
antibody feed steam (30 mg/mL), and/or on the buffer-exchanged
antibody in conjugation reaction buffer. The FlowVPE or UV sensor
can be used to measure the concentration of the antibody in the
different feed steams. Additionally, flow meters on all of the feed
steams can be used to monitor and adjust the flow rate of any
component to ensure the buffer-exchange in the ILDF is occurring at
30 mg/mL. Furthermore, a conductivity or pH probe after the ILDF
can be used to ensure that the buffer-exchange of the antibody into
the conjugation reaction buffer was complete.
[0186] After the conjugation, conventional TFF2 is typically
performed in three steps which all occur in a single unit
operation. The post-conjugation crude reaction mixture is first
concentrated via ultrafiltration to 30 mg/mL (from 15 mg/mL
post-conjugation reaction). The conjugate is then diafiltered for 4
diavolumes against conjugation reaction buffer in what is referred
to as Stage I of TFF2. During Stage I, residual impurities are
removed from the crude-reaction mixture. Directly after Stage I,
Stage II is performed by diafiltering the conjugate against 8
diavolumes of basal formulation buffer. Stage II is designed as
strictly a buffer-exchange step but does contribute to additional
clearance of residual impurities.
[0187] For a continuous process utilizing SPTFF, three separate
steps were evaluated. First, the ILC module for concentrating was
used. The ILC followed the conjugation reaction tubing. In a single
pass, the conjugate entering the ILC at 15 mg/mL was concentrated
to 30 mg/mL with the volume removed being diverted to waste. The
concentration factor used for IMGN853 and described in this
instance was two (2), but other concentration factors could be used
in place to achieve the same result of concentrating the conjugate
before diafiltration.
[0188] After the ILC, the 30 mg/mL conjugate feed stream was fed
into the first ILDF (ILDF1) which was meant to emulate Stage I. The
diafiltration buffer feed for ILDF1 was conjugation reaction
buffer, and it was added at the appropriate volume to achieve the
desired number of diavolumes in a single pass. For IMGN853, Stage I
was achieved in four conventional diavolumes. Proof of concept
studies evaluated the removal of residual impurities as a function
of number of diavolumes by adjusting the flow rate of the
diafiltration buffer feed pump.
[0189] After ILDF1, the conjugate remained at 30 mg/mL but had
significantly lower concentrations of impurities present. The exit
stream from ILDF1 fed ILDF2 where the diafiltration buffer was
basal formulation buffer. The conjugate entered ILDF at 30 mg/mL in
conjugation reaction buffer (pH 7.6 to 8.7) and exited the ILDF2 at
30 mg/mL in pH 5.0 buffer.
[0190] Initial proof of concept studies performed using Pall Life
Sciences ILDF modules used a T01 module which has a membrane area
of 0.11 m.sup.2. The module was evaluated at flow rates between 1-4
mL/min for the conjugate feed. The diafiltration buffer feed rates
were varied such that the number of diavolumes varied. Processing
of between one and thirteen diavolumes were evaluated using the
ILDF. For all of the studies, conjugate material was generated
using conventional batch processing and frozen into aliquots. Free
maytansinoid species were quantified prior to freezing and again
after thawing (prior to processing). The crude-reaction mixture was
fed into the ILC. The flow rate of the retentate stream was
measured using a flow meter to calculate the concentration factor
achieved over the module. The IMGN853 process and all studies were
performed using a target concentration factor of two.
[0191] A first study with ILDF was conducted to evaluate clearance
of residual impurities from IMGN853 conjugation reaction. A bulk
conjugation was performed to generate material for the study using
target reaction conditions. The post-conjugation crude reaction
mixture was filtered, aliquotted, and frozen prior to use. After
thawing, the crude reaction mixture was concentrated using the
single pass ILC module targeting a concentration factor of two. The
crude reaction mixture was at 15 mg/mL, and the target retentate
concentration of the conjugate post-ILC was 30 mg/mL. The post-ILC
conjugate was then diluted to 10 mg/mL using conjugation reaction
buffer to test the ILDF module at a low starting concentration. The
dilution from 30 mg/mL to 10 mg/mL prior to the ILDF effectively
reduced the load of free drug impurities in the conjugate feed.
This is evident in FIG. 6 showing free drug impurity levels for the
four main impurity species before and after the ILC. The
post-conjugation crude reaction mixture has high impurity levels.
After concentrating over the ILC and then diluting to 10 mg/mL, the
concentration of impurities were reduced to approximately 1/3 of
the starting concentration.
[0192] The 10 mg/mL conjugate was fed to the T01 ILDF module at 4
mL/min targeting 7 diavolumes of processing for stage I. The Stage
I diafiltration buffer was conjugation reaction buffer (15 mM
potassium phosphate, 2 mM EDTA, pH 8.7). Samples were taken from
the retentate line and analyzed for protein concentration to
determine when the conjugate eluted from the system. After the
concentration reached a steady-state, samples were collected from
the retentate line and analyzed by RP-HPLC to quantify free drug
impurity levels and calculate clearance over the ILDF. FIG. 6 shows
the results for three samples taken from the ILDF1 step: early,
late, and pooled. The early sample was taken shortly after
steady-state was achieved on the ILDF. The impurity levels in all
three samples were well below specification limits. The reduction
in free drug levels over the ILDF was significant and acceptable in
terms of process performance.
[0193] After the first pass over the ILDF against conjugation
reaction buffer to mimic Stage I of TFF2, the resulting pooled
conjugate was then passed through the module again to emulate Stage
II. Prior to Stage II, the module was flushed and then prepped for
diafiltration against basal formulation buffer (10 mM acetate, 9%
sucrose, pH 5.0). The conjugate was fed at 4 mL/min and the
diafiltration buffer feed flow rate was set to target 13 diavolumes
across the ILDF. This number was chosen to match eight diavolumes
for conventional TFF processing.
[0194] Similar to ILDF1, samples were collected from the retentate
line and analyzed to measure concentration of the antibody. When
the protein concentration was detected, the product was collected
and samples were taken periodically from the retentate line. After
the system reached steady-stage, the samples collected from the
retentate line were analyzed by RP-HPLC to quantify residual
impurity levels and to calculate clearance over ILDF2. After the
product had been passed over the ILDF and collected, the pooled
material was filtered and then analyzed by SEC and RP-HPLC. The
results for all samples from ILDF2, emulating the Stage II
buffer-exchange during conventional TFF2 processing, are shown in
FIG. 6. Free drug levels were all <1% which is well below the
final specification for purified drug substance (DS). These results
demonstrate continuous purification of IMGN853 conjugate by SPTFF
is feasible and capable of generating purified conjugate with
acceptable product quality as measured by SEC and RP-HPLC.
[0195] The next study examined the clearance capacity of the ILDF
to remove residual impurities at a feed concentration of 15 mg/mL.
This concentration was chosen after the first study showed adequate
clearance at 10 mg/mL. During this study, the conjugate was not
processed by the ILC. Crude conjugate reaction mixture from a bulk
conjugation was thawed and fed directly to the ILDF. A T01 ILDF
module was used and the feed flow rate was 4 mL/min. Conjugation
reaction buffer was used for the diafiltration buffer and the feed
flow rate was set to initially target one diavolume. When the
conjugate was detected in the retentate by measuring concentration,
samples were collected from the retentate line and analyzed by
RP-HPLC to quantify free drug levels. The results are shown in FIG.
7. The post-conjugation crude reaction mixture had high starting
levels that were in line with previous conjugations. After a single
pass equivalent to one diavolume of processing, the total free drug
levels dropped from approximately 26% in the feed to 7% in the
retentate.
[0196] The diafiltration feed flow rate was then increased to
target seven diavolumes, which is comparable to the target four
diavolumes for conventional TFF processing. After the system
reached steady-state, the free drug levels were measured in the
retentate. As shown in FIG. 4, the free drug levels dropped
significantly after a single pass under these processing
conditions. The final total free drug levels were below 1%, which
is within the acceptance range for final product quality. These
results indicate the ILDF performs well at 15 mg/mL feed conjugate
concentration and removes impurities to levels below specification
limits when processed for seven diavolumes.
[0197] For comparison, free drug impurity clearance values from a
batch TFF2 purification process are shown in FIG. 8. After 4
diavolumes of conventional TFF2 batch processing (solid bars), the
total free drug impurity level was 3.4%. After 7 diavolumes of
SPTFF processing (striped bars), which is equivalent to 4
conventional diavolumes, the total free drug level was 0.7%. One
important difference between these two studies was the
concentration of the conjugate during purification. For the
continuous processing study, the conjugate feed to the ILDF module
was 15 mg/mL. The conventional batch TFF processing was performed
at 30 mg/mL.
[0198] The ILDF performed well at 15 mg/mL with no concerns of
pressure or product quality, so the conjugate concentration of the
feed was increased to optimize yield from the ILDF in a follow up
study. The target feed conjugate concentration was 30 mg/mL. Crude
reaction mixture was first passed through the ILC targeting a
concentration factor of two. The conjugate entered the ILC at 15
mg/mL and was concentrated to a target of 30 mg/mL. The resulting
conjugate at 30 mg/mL was collected before feeding to the ILDF. The
diafiltration buffer for the ILDF was conjugation reaction buffer,
and the feed flow rate was set to target seven diavolumes. The feed
flow rate for the conjugate at 30 mg/mL was 4 mL/min on a T01 ILDF
module. After protein was detected in the retentate stream, the
conjugate was collected and samples were collected. The system was
allowed to reach a steady-state before a sample was analyzed by
RP-HPLC to quantify free drug levels and to calculate clearance
over the ILDF. The results are shown in FIG. 9. The post-ILDF free
drug levels were compared to samples taken prior to the ILC (15
mg/mL of crude reaction mixture) and post-ILC (30 mg/mL). The
post-ILC free drug levels increased slightly which was attributed
to slightly overconcentrating the conjugate. The post-ILC conjugate
was measured at 31 mg/mL indicating a concentration factor of 2.1
was used. The resulting free drug levels were slightly higher, but
to accurately assess the ILDF, the conjugate was diluted to 30
mg/mL by adding the appropriate volume of conjugation reaction
buffer to the pooled material prior to feeding the ILDF.
[0199] The sample collected and analyzed by RP-HPLC from the ILDF
at 4 mL/min showed clearance of free drug species from the
pre-ILDF/post-ILC sample. However, the concentration of conjugate
in the retentate was measured at approximately 20 mg/mL. It was
posited that the drop in conjugate concentration was attributed to
aggregation and accumulation of product on the membrane in the
cassette. To alleviate any clogging and reduce the pressure in the
system, the feed flow rate was reduced to 3 mL/min. Accordingly,
the diafiltration buffer feed pump flow rate was also decreased
such that seven diavolumes was targeted for processing. After
reducing the inlet flow rates for the ILDF, a sample was collected
from the retentate and analyzed for concentration and free drug
levels. The concentration of the sample was determined to be 22.5
mg/mL, and the free drug levels were comparable to the sample
measured at 4 mL/min. Collectively, this indicates 7 diavolumes of
processing is sufficient to purify the conjugate but a maximum of
23 mg/mL conjugate concentration in the retentate was observed.
Example 2: A Continuous Conjugation Process for the Conjugation of
IMGN632
[0200] The continuous conjugation process for the conjugation of
the ADC IMGN632 is detailed in flow diagram represented in FIG. 10.
IMGN632 contains an anti-CD123 antibody ("G4732A") linked to the
cytotoxic payload DGN549-C. IMGN632 is shown in FIG. 11.
Example 2A: Flow Chemistry Studies
[0201] The reaction set up is similar to the set up shown in FIG.
5. The first syringe contains the DNA-alkylating IGN payload
"DGN549C" dissolved in DMA. The second syringe contains bisulfite
stock solution and 50 mM succinate pH 3.3 at appropriate volumetric
ratios. Both of these feed streams mix in the static in-line mixer
to initiate the sulfonation reaction. The DGN549C targets a final
concentration of 1 mM in the mixed stream. The resulting mixture is
50 mM succinate pH 3.3 and 50% DMA with a 1.4 molar excess of
bisulfite to DGN ratio. The two feed streams exit the static
in-line mixer in a single stream. The length of the tubing is
determined based on volumetric flow rates such that the residence
time of the reactants in the tubing targets 3 hours. The
temperature of the sulfonation reaction is 25.degree. C. and is
maintained by submerging the tubing into a temperature controlled
water bath. The third syringe (Ab+Buffer syringe from FIG. 5),
contains reoxidized anti-CD123 antibody ("G4723") that has been
adjusted to pH 6.0 with 5% succinic acid. Appropriate volumes of
DMA and 50 mM potassium phosphate are added to the reoxidized
antibody mixture. The homogeneous mixture is drawn up in a single
syringe to be combined in-line with the sulfonation reaction.
[0202] The sulfonation feed stream enters a second static mixer
where it mixes with the pH adjusted reoxidized antibody. The
volumetric flow rates are adjusted such that the final conjugation
stream exiting the second static mixer achieves the desired
reaction conditions of 2.0 mg/mL antibody concentration, 15% DMA,
and a 3.5 molar ratio of DGN to antibody. The conjugate tubing coil
length is chosen such that the residence time targets a conjugation
reaction duration of 20 hours. Similar to the sulfonation tubing,
the conjugation tubing is submerged in a temperature controlled
water bath set at 25.degree. C.
Example 2B: Single-Pass Tangential Flow Filtration Studies
[0203] The pH adjusted crude conjugate mixture at pH 4.2 is fed
into the ILC at a concentration of 2 mg/mL targeting a
concentration factor of four so that the retentate is approximately
8 mg/mL. The resulting conjugate material at 8 mg/mL is fed to an
ILDF SPTFF module. The diafiltration buffer for the first ILDF is
10 mM succinate, 50 .mu.M bisulfite pH 4.2 with 10% (v/v) DMA. The
flow rates of the ILDF are adjusted to target 7 diavolumes,
matching the four diavolumes used during conventional batch
processing.
[0204] After the first ILDF, the material is fed at the same
concentration to a second ILDF module where the diafiltration
buffer is 10 mM succinate 50 .mu.M bisulfite pH 4.2. The flow rates
of the diafiltration buffer feed and product feed are set such that
the target number of diavolumes is 15 to achieve a similar
buffer-exchange to the 10 diavolumes targeted during conventional
batch TFF processing.
Example 3: Pulse Treatment Improves Conjugation of IMGN853
[0205] The present inventors have discovered that continuous flow
conjugation allows for a pulsing change of a parameter (e.g.,
temperature or pH). The pulsing is possible in a continuous flow
conjugation process where reactions take place as reagents pass
through coils and tubing with small volumes. For example, short
sections of the coils or tubing can be rapidly heated (e.g., by
insulation) or cooled to create a short temperature excursion or
"pulse." (See e.g., FIG. 12).
[0206] The inventors have further discovered that pulsing changes
in reaction parameters is advantageous, for example, where longer
exposure to the altered parameter may damage the process or result
in negative impacts to product quality. For instance, increasing
the temperature of a conjugation reaction for an extended period of
time can result in increased aggregation. In contrast, as
demonstrated herein, increasing the temperature of a conjugation
reaction in only short pulses of time can increase reaction speed
without significantly increasing aggregate formation.
[0207] Conjugation of IMGN853 was performed in vials with
temperature pulsing, which involved placing a vial containing the
conjugation reaction components at 20.degree. C. for about 30
minutes and then pulsing by placing the vial into a water bath at a
higher temperature for a specified pulse time and then returning
the vial to 20.degree. C. The vial recovered at 20.degree. C. until
the next pulse occurred, and the process was repeated for a
specified number of pulses. Four different pulse conditions were
assessed: 4 pulses at 40.degree. C. for 7 minutes each; 4 pulses at
42.5.degree. C. for 7 minutes each; 5 pulses at 50.degree. C. for 1
minute each, and 5 pulses at 60.degree. C. for 1 minute each. A
control reaction was performed with a steady 20.degree. C.
temperature.
[0208] The extent of reaction completion (%) was used to determine
the reaction rate. At a steady 20.degree. C. temperature, an
IMGN853 conjugation reaction requires about 8 hours to reach
completion. (See FIG. 14, left panel). All pulse conditions tested
decreased reaction time (i.e., reach 100% completion in less time).
(See FIG. 14, left panel). Pulsing (5 times) at 60.degree. C. for 1
minute per pulse dramatically increased the occurrence of high
molecular weigh (HMW; aggregate) species. (See FIG. 14, right
panel). In contrast, all of the other pulsing conditions tested had
little impact on the occurrence of HWM (aggregate) species.
[0209] This data demonstrate that reaction conditions enabled by
continuous flow conjugation can improve reaction kinetics and
productivity without compromising product quality.
Example 4: Single-Step Preparation of Antibody for Conjugation
[0210] In Example 2, the IMGN632 drug substance process involves
two steps to prepare the G4723A antibody for conjugation: reduction
and reoxidation (FIG. 10, top left box). The reduction of the
G4723A antibody reduces interchain disulfides between native
cysteines and removes capping groups on the C442 engineered
cysteine residues. This step involves addition of 25 molar
equivalents of TCEP to the G4723A antibody. After the reduction
reaction, the reduced antibody mixture is buffer-exchanged to
remove any residual TCEP and prepare the antibody for the
reoxidation reaction. The reoxidation step involves addition of 8
molar equivalents of DHAA to reduced G4723A antibody to reform the
interchain disulfides of the antibody. The reaction leaves the C442
engineered cysteines residues as free thiols that can be conjugated
to the payload via a maleimide function group.
[0211] To demonstrate the feasibility of performing these reactions
in a continuous format, small-scale studies were performed using a
micro-scale flow reactor.
Example 4A: Reduction Reaction
[0212] The reduction reaction was performed to demonstrate that the
formulated G4723A antibody could be reduced in a flow reactor and
yield antibody with consistent product quality as a side-by-side
batch reaction.
[0213] A 150 mg reduction scale based on G4723A antibody was
performed to demonstrate continuous reduction in a flow reactor.
The formulated G4723A antibody at 51.1 mg/mL was used as one feed
stream. The antibody solution was drawn into a syringe. The
TCEP-HCl stock solution, prepared at 100 mM in 50 mM potassium
phosphate, pH 7.5 was combined with additional 50 mM potassium
phosphate, pH 7.5 as the second feed stream for the continuous
reaction. This was done due to the low flow rates and volumetric
ratios of TCEP-HCl stock solution to the 50 mM potassium phosphate,
pH 7.5 buffer feed streams if they were kept separate. The
additional 50 mM potassium phosphate, pH 7.5 buffer is required to
dilute the formulated G4723A antibody to 5 mg/mL for the reduction
reaction.
[0214] The reaction was setup using Harvard Apparatus syringe pumps
and 1/16.sup.th PEEK tubing. The volumetric ratio of the two feed
streams were chosen such that the final reaction stoichiometry
after mixing was at the target reaction conditions--25 molar
equivalents of TCEP-HCl to antibody, 5 mg/mL antibody. The combined
volumetric flow rate of the two feed streams was used to calculate
the length of PEEK tubing required so that the reaction duration,
or residence time in the flow reactor, was 16 hours. For this
experiment, both the side-by-side batch control and the continuous
reactions were performed at ambient temperature.
[0215] A side-by-side batch control reduction reaction was setup at
a 20 mg G4723A antibody scale. All reaction parameters were
matched. After the 16 hour reaction duration, the control and
continuous reactions were sampled and labeled for analysis. The
continuous reduction reaction was sampled from the outlet of the
flow reactor. The bulk control batch reaction was sampled for
analysis.
[0216] To characterize the reduction reaction, the samples were
labeled with Alexa-MAL 488 fluorophore, which reacts with free
thiols on the antibody and can be detected by UV spectrometry. The
samples were reacted with the Alexa-MAL 488 and analyzed by SE-HPLC
to calculate the Alexa-Antibody ratio. The results for the
continuous and side-by-side control reactions are shown in Table
6.
TABLE-US-00007 TABLE 6 Batch vs. Continuous Reduction Reaction
Sample Alexa-Antibody Ratio Alexa-Labeled, Reduced (Control) 7.50
Alexa-Labeled, Reduced (Continuous) 7.92
[0217] The data shown in Table 6 indicate the G4723A antibody was
reduced by traditional batch and continuous processing. For the
formulated G4723A antibody prior to reduction, no Alexa labeling
has been observed. After the interchain disulfides are reduced to
form free thiols during the reduction reaction, the Alexa-MAL 488
is able to react with and conjugate the free thiol. Complete
reduction of the engineered cysteines and native cysteines of the
antibody would result in 10 labels.
[0218] Approximately 7-8 Alexa-MAL 488 labels were detected in both
samples, indicating the G4723A antibody was reduced. These results
were consistent with previous development studies on smaller scale
where 8 Alexa-MAL 488 labels are identified after the reduction
reaction. The difference from the theoretical target of 10 labels
is likely due to some reoxidation that occurs on small scale,
driven by air in the reaction vessel, causing free thiols to reform
disulfides and decrease the number of available free thiols on the
antibody for conjugation with Alexa-MAL 488.
[0219] Subsequent studies were repeated on larger scales and
targeting shorter reduction times of approximately 3 hours.
Similarly, the antibodies generated from these studies were labeled
with Alexa-MAL 488 and characterized by SE-HPLC to compare the
reaction efficiency. The product quality data from a subsequent
study in which the reduction reaction time was targeted for 3 hours
by increasing the volumetric flow rates of the two feed stream are
shown in Table 7.
TABLE-US-00008 TABLE 7 Product Quality Evaluation of Continuous
Reduction Reaction Alexa:Ab HMW Monomer Sample Ratio (%, SEC) (%,
by SEC) Control_post- 8.50 0.57 99.17 reduction Continuous_post-
8.14 0.11 99.58 reduction
[0220] In addition to the Alexa:Ab ratio comparing the extent of
the reduction reaction for the batch control and continuous
reaction, the Monomer and HMW values for the two reaction products
were measured. The data in Table 7 show that at a larger scale with
higher volumetric flow rates, a continuous reduction reaction is
comparable to the batch control. Additionally, there is a slight
increase in the Monomer for the antibody produced by the continuous
reaction.
[0221] These results demonstrate that the reduction reaction can be
successfully performed in a continuous format using flow
chemistry.
Example 4B: Reoxidation Reaction
[0222] The reoxidation reaction was performed to demonstrate the
reduced G4723A antibody can be reoxidized in a continuous
format.
[0223] Due to the scale of the reactions and flow reactor
equipment, the reduced antibodies used to supply the reoxidation
studies were NAP purified. During IMGN632 manufacturing, the
reduced antibody is TFF purified to remove any residual TCEP-HCl
from the reaction solution. After the TCEP-HCl is removed, DHAA is
added to the reaction pool to reform the interchain disulfides of
the antibody while leaving the two engineered C442 cysteine
residues free for conjugation.
[0224] For the reoxidation studies, the G4723A antibody was first
reduced using a batch reaction because of the need to setup the
flow reactor equipment for the reoxidation reaction conditions. The
reduction reaction was performed at 37.degree. C. for 1 hour to
completely reduce the antibody using the same 25 molar equivalents
of TCEP-HCl relative to antibody at an antibody concentration of 5
mg/mL. After the reduction reaction, a sample of the reaction was
Alexa-labeled to confirm the reduction of the antibody.
[0225] The reduced antibody was then NAP purified to remove
residual TCEP-HCl from the solution prior to the addition of DHAA.
The NAP purified material was measured for concentration to ensure
the reoxidation reaction setup targeted the appropriate
stoichiometric ratios. The NAP purified reduced antibody was split
so that a side-by-side batch control reaction could be setup as a
control with the continuous reoxidation sample.
[0226] For the continuous reoxidation reaction, three feed streams
were used. The first was the reduced antibody that was NAP
purified. The second was the 100 mM DHAA stock solution in DMA
combined with additional DMA as required to target the final 5%
(v/v) target for the reaction. The third was the 50 mM potasasium
phosphate pH 7.5 buffer that dilutes the NAP purified reduced
antibody to the target 2.5 mg/mL antibody concentration for the
reaction. The volumetric ratios of these three feed streams were
determined based on the NAP purified reduced antibody
concentration. The ratios were adjusted so that the combined feed
streams gave the final target reoxidation reaction conditions after
mixing. The volumetric flow rates were also used to calculate the
amount of 1/16.sup.th PEEK tubing required to target a reaction
duration of 6 hours. The side-by-side reoxidation reaction and
continuous reoxidation reaction were both setup at ambient
temperature.
[0227] A 50 mg reoxidation reaction was setup for the continuous
and control reactions. A total of 19.9 feet of 1/16.sup.th PEEK
tubing was required to target 6 hours based on the volumretric flow
rates of the three feed streams. The continuous reoxidation
reaction was sampled intermittently from the outlet of the flow
reactor starting at 6.5 hours after the start of the reaction. The
additional time was given to ensure the reaction reached
steady-state based on potential backpressure within the flow
reactor. Samples taken from the outlet of the flow reactor or from
the bulk of the batch reoxidation reaction were all labeled with
Alexa-MAL 488 fluorophore and characterized by SE-HPLC. The
reoxidation reaction was sampled over time to ensure a steady-state
was achieved. Additionally, the reduced antibody that was contained
within the syringe and fed as one of the feed streams for the
continuous reoxidation reaction was also labeled after the end of
the study. This sample was tested to ensure the reduced antibody
did not reoxidize while sitting in the syringe over the duration of
the study. This sample was compared with the sample after reduction
to understand any changes in the reduced antibody potentially
caused by ambient conditions. The results of this continuous
reoxidation study are shown in Table 8.
TABLE-US-00009 TABLE 8 Continuous Reoxidation Reaction Product
Quality Sample Alexa:Ab Ratio NAP Purified, Post-Reduction TO 8.15
Alexa-Labeled, Continuous Reoxidized (T = 7 h) 2.37 Alexa-Labeled,
Continuous Reoxidized (T = 8 h) 2.36 Continuous Reoxidized (T = 9
h) 2.35 Control Reoxidized 2.22 Reduced Antibody from Syringe (End)
7.36
[0228] These data indicate that the reoxidation reaction can be
successfully performed using continuous processing. The reoxidized
reaction performed in the flow reactor exhibited successful
reoxidation with approximately 2.4 Alexa-MAL 488 labels. The data
from the batch control reaction was comparable with approximately
2.2 Alexa-MAL 488 labels. The reduced antibody prior to reoxidation
had approximately 8 labels which demonstrates the addition of the
DHAA caused the reformation of disulfides. The remaining labels
that were conjugated with Alexa-MAL 488 are the engineered cysteine
residues that would be available for conjugation. Additionally, the
data shown in Table 8 indicate the reduced antibody undergoes very
minimal reoxidation under ambient conditions. The reduced antibody
started with 8.15 labels and after the duration of the reoxidation
study only decreased to 7.36. This data indicates the reoxidation
was driven by the reaction with DHAA and not by air present in the
reaction vessel or buffers.
[0229] Collectively, these results show the reoxidation reaction
can be successfully achieved using continuous processing in a flow
reactor.
[0230] It is to be appreciated that the Detailed Description
section, and not the Summary and Abstract sections, is intended to
be used to interpret the claims. The Summary and Abstract sections
may set forth one or more but not all exemplary embodiments of the
present invention as contemplated by the inventor(s), and thus, are
not intended to limit the present invention and the appended claims
in any way.
[0231] The present invention has been described above with the aid
of functional building blocks illustrating the implementation of
specified functions and relationships thereof. The boundaries of
these functional building blocks have been arbitrarily defined
herein for the convenience of the description. Alternate boundaries
can be defined so long as the specified functions and relationships
thereof are appropriately performed.
[0232] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present invention. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
[0233] The breadth and scope of the present invention should not be
limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
[0234] The claims in the instant application are different than
those of the parent application or other related applications. The
Applicant therefore rescinds any disclaimer of claim scope made in
the parent application or any predecessor application in relation
to the instant application. The Examiner is therefore advised that
any such previous disclaimer and the cited references that it was
made to avoid, may need to be revisited. Further, the Examiner is
also reminded that any disclaimer made in the instant application
should not be read into or against the parent application.
Sequence CWU 1
1
5615PRTArtificial SequencehuMov19 VH CDR1 1Gly Tyr Phe Met Asn1
5217PRTArtificial SequencehuMov19 VH CDR2 2Arg Ile His Pro Tyr Asp
Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln1 5 10 15Gly39PRTArtificial
SequencehuMov19 VH CDR3 3Tyr Asp Gly Ser Arg Ala Met Asp Tyr1
5415PRTArtificial SequencehuMov19 VL CDR1 4Lys Ala Ser Gln Ser Val
Ser Phe Ala Gly Thr Ser Leu Met His1 5 10 1557PRTArtificial
SequencehuMov19 VL CDR2 5Arg Ala Ser Asn Leu Glu Ala1
569PRTArtificial SequencehuMov19 VL CDR3 6Gln Gln Ser Arg Glu Tyr
Pro Tyr Thr1 575PRTArtificial SequenceZ4681A VH CDR1 7Ser Tyr Tyr
Ile His1 5817PRTArtificial SequenceZ4681A VH CDR2 8Val Ile Tyr Pro
Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln1 5 10
15Gly99PRTArtificial SequenceZ4681A VH CDR3 9Glu Val Arg Leu Arg
Tyr Phe Asp Val1 51017PRTArtificial SequenceZ4681A VL CDR1 10Lys
Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu1 5 10
15Ala117PRTArtificial SequenceZ4681A VL CDR2 11Trp Ala Ser Thr Arg
Glu Ser1 5128PRTArtificial SequenceZ4681A VL CDR3 12His Gln Tyr Leu
Ser Ser Arg Thr1 5135PRTArtificial SequenceG4723A VH CDR1 13Ser Ser
Ile Met His1 51417PRTArtificial SequenceG4723A VH CDR2 14Tyr Ile
Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10
15Gly1512PRTArtificial SequenceG4723A VH CDR3 15Glu Gly Gly Asn Asp
Tyr Tyr Asp Thr Met Asp Tyr1 5 101611PRTArtificial SequenceG4723A
VL CDR1 16Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser1 5
10177PRTArtificial SequenceG4723A VL CDR2 17Arg Val Asn Arg Leu Val
Asp1 5189PRTArtificial SequenceG4723A VL CDR3 18Leu Gln Tyr Asp Ala
Phe Pro Tyr Thr1 5195PRTArtificial SequencehuCMET-27 VH CDR1 19Ser
Tyr Asp Met Ser1 52017PRTArtificial SequencehuCMET-27 VH CDR2 20Thr
Ile Asn Ser Asn Gly Val Ser Ile Tyr Tyr Pro Asp Ser Val Lys1 5 10
15Gly219PRTArtificial SequencehuCMET-27 VH CDR3 21Glu Glu Ile Thr
Thr Glu Met Asp Tyr1 52215PRTArtificial SequencehuCMET-27 VL CDR1
22Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Ile His1 5 10
15237PRTArtificial SequencehuCMET-27 VL CDR2 23Arg Ala Ser Asn Leu
Glu Ser1 5249PRTArtificial SequencehuCMET-27 VL CDR3 24Gln Gln Ser
Asn Glu Glu Pro Leu Thr1 5255PRTArtificial SequencehuB4 VH CDR1
25Ser Asn Trp Met His1 52610PRTArtificial SequencehuB4 VH CDR2
26Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn1 5 102711PRTArtificial
SequencehuB4 VH CDR3 27Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr1
5 102810PRTArtificial SequencehuB4 VL CDR1 28Ser Ala Ser Ser Gly
Val Asn Tyr Met His1 5 10297PRTArtificial SequencehuB4 VL CDR2
29Asp Thr Ser Lys Leu Ala Ser1 5307PRTArtificial SequencehuB4 VL
CDR3 30His Gln Arg Gly Ser Tyr Thr1 5315PRTArtificial
SequencehuADAM9 VH CDR1 31Ser Tyr Trp Met His1 53217PRTArtificial
SequencehuADAM9 VH CDR2 32Glu Ile Ile Pro Ile Phe Gly His Thr Asn
Tyr Asn Glu Lys Phe Lys1 5 10 15Ser3314PRTArtificial
SequencehuADAM9 VH CDR3 33Gly Gly Tyr Tyr Tyr Tyr Phe Asn Ser Gly
Thr Leu Asp Tyr1 5 103415PRTArtificial SequencehuADAM9 VL CDR1
34Lys Ala Ser Gln Ser Val Asp Tyr Ser Gly Asp Ser Tyr Met Asn1 5 10
15357PRTArtificial SequencehuADAM9 VL CDR2 35Ala Ala Ser Asp Leu
Glu Ser1 5369PRTArtificial SequencehuADAM9 VL CDR3 36Gln Gln Ser
His Glu Asp Pro Phe Thr1 537118PRTArtificial SequencehuMov19 VH
37Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu
Trp Ile 35 40 45Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn
Gln Lys Phe 50 55 60Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Asn Thr Ala His65 70 75 80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp
Phe Ala Val Tyr Tyr Cys 85 90 95Thr Arg Tyr Asp Gly Ser Arg Ala Met
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11538112PRTArtificial SequencehuMov19 VL version 1.00 38Asp Ile Val
Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Pro
Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30Gly
Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40
45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp
50 55 60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile
Ser65 70 75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Ser Arg 85 90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 11039112PRTArtificial SequencehuMov19 VL
version 1.60 39Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val
Ser Leu Gly1 5 10 15Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser
Val Ser Phe Ala 20 25 30Gly Thr Ser Leu Met His Trp Tyr His Gln Lys
Pro Gly Gln Gln Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu
Glu Ala Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Lys Thr
Asp Phe Thr Leu Thr Ile Ser65 70 75 80Pro Val Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95Glu Tyr Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
11040118PRTArtificial SequenceZ4681A VH 40Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Ile His Trp
Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 35 40 45Gly Val Ile
Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 50 55 60Gln Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser 11541113PRTArtificial
SequenceZ4681A VL 41Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala
Val Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln
Ser Val Phe Phe Ser 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr
Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro Arg Leu Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Pro
Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95Tyr Leu Ser Ser Arg
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg42121PRTArtificial SequenceG4723A VH 42Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser 20 25 30Ile Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr
Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys
Gly Arg Ala Thr Leu Thr Ser Asp Arg Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Gly Gly Asn Asp Tyr Tyr Asp Thr Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12043108PRTArtificial SequenceG4723A VL 43Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu Ser Trp Phe
Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45Tyr Arg Val
Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ala Phe Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10544118PRTArtificial SequencehuCMET-27 VH 44Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Asn Ser Asn Gly Val Ser Ile Tyr Tyr Pro Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Glu Ile Thr Thr Glu Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11545112PRTArtificial
SequencehuCMET-27 VL 45Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Glu Ser Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Ile His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser
Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Glu Pro Leu
Thr Phe Gly Gln Gly Thr Lys Val Glu Leu Lys Arg 100 105
11046120PRTArtificial SequencehuB4 VH 46Gln Val Gln Leu Val Gln Pro
Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys
Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30Trp Met His Trp Val
Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp
Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Asn Phe 50 55 60Gln Gly Lys
Ala Lys Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Ser Val Thr Val Ser Ser 115
12047105PRTArtificial SequencehuB4 VL 47Glu Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Thr
Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20 25 30His Trp Tyr Gln Gln
Lys Pro Gly Thr Ser Pro Arg Arg Trp Ile Tyr 35 40 45Asp Thr Ser Lys
Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly
Thr Asp Tyr Ser Leu Thr Ile Ser Ser Met Glu Pro Glu65 70 75 80Asp
Ala Ala Thr Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90
95Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 10548123PRTArtificial
SequencehuADAM9 VH 48Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile Ile Pro Ile Phe Gly
His Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Arg Phe Thr Ile Ser
Leu Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Gly Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly
Tyr Tyr Tyr Tyr Phe Asn Ser Gly Thr Leu Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 12049111PRTArtificial
SequencehuADAM9 VL 49Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Ser Cys Lys Ala Ser
Gln Ser Val Asp Tyr Ser 20 25 30Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Asp Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95Glu Asp Pro Phe
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
11050447PRTArtificial SequencehuMov19 Heavy 50Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Phe Met Asn
Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45Gly Arg
Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60Gln
Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His65 70 75
80Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys
85 90 95Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44551218PRTArtificial SequencehuMov19 Light 1.00 51Asp Ile Val Leu
Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Pro Ala
Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30Gly Thr
Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45Arg
Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55
60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Asn Ile Ser65
70 75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser
Arg 85 90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21552218PRTArtificial SequencehuMov19 Light 1.60 52Asp Ile Val Leu
Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Pro Ala
Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30Gly Thr
Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45Arg
Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro Asp 50 55
60Arg Phe Ser Gly Ser Gly Ser Lys Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Pro Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser
Arg 85 90 95Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21553447PRTArtificial SequenceZ4681A Heavy 53Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Ile His
Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 35 40 45Gly Val
Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 50 55 60Gln
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44554219PRTArtificial SequenceZ4681A Light 54Glu Ile Val Leu Thr
Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Arg Val Thr
Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30Ser Ser Gln
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro
Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln
85 90 95Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200
205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21555450PRTArtificial SequenceG4723A Heavy 55Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Ser 20 25 30Ile Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr
Ile Lys Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys
Gly Arg Ala Thr Leu Thr Ser Asp Arg Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Gly Gly Asn Asp Tyr Tyr Asp Thr Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Cys Leu Ser 435 440
445Pro Gly 45056214PRTArtificial SequenceG4723A Light 56Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu
Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40
45Tyr Arg Val Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ala
Phe Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys 210
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