mAbChem poster-2015 ADC meeting

Process Development of ADCs in mAbChem Rongliang Lou, Dev Sharma, Daniel Wang, Ping Ge

23 Business Park Drive, Branford, CT 06405 [email protected] www.mabchemlab.com

World ADC Summit, San Diego, Oct 19-22th, 2015

Abstract Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies

(selectivity) and cytotoxic small molecules (potency) for targeted delivery to cancer cells

thereby enhancing their antitumor activity and minimizing off target toxicity. Preparation of

ADCs with desired profile is a key step in ADC development. Attaching a toxin or

payload to an antibody can be accomplished through a variety of conjugation approaches,

conventionally via lysine residues utilizing amide bonds (as in Kadcyla) or cysteine residues

utilizing thiother bonds (as in Adcetris), or more recently a number of site directed protocols

utilizing a variety of reliable chemistries.

The concept of ADCs appears simple, however, the development of efficacious, safe and

reproducible product presents a considerable challenge. In this poster, we would like to share

our experience in the process development of ADCs via conventional cysteine-mediated

conjugation. Several parameters involved in the bioconjugation including reaction time,

scale, pH, stoichiometry, etc have been investigated. Conjugates with different drug loading

were also separated as individual species from the crude mixture by using hydrophobic

interaction chromatography (HIC).

Introduction ADC Drugs on the market

Kadcyla: conventional lysine conjugated ADC, used for treatment of HER2 positive

metastatic breast cancer

Adcetris: conventional cysteine conjugated ADC, used for treatment of Hodgkin’s

lymphoma or systemic anaplastic large cell lymphoma

Methods used to prepare ADCs

Lysine mediated conjugation: conjugation of the drugs to lysine residues scattered

throughout the entire mAb structure, leading to a multitude of species, many of which

may display undesirable physico-chemical and biological characteristics

Cysteine mediated conjugation: involving the use of endogenous interchain cysteine

residues for conjugation with maleimide-containing linker-payloads

Site specific conjugation: using engineered mAbs designed for conjugation at specific

cysteine residues or other functional groups

Enzymatic based conjugations: conjugation to specific tags/residues on mAbs utilizing

enzyme

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Multiple ADC Architectures

Lysine mediated

conjugation

Cysteine mediated

Interchain disulfide

conjugation

Site specific

conjugation

%

Drug loading

% %

Drug loading Drug loading

Advantages of Cysteine Mediated Conjugation

Less number of conjugated species with simpler HIC than lysine mediated conjugates;

i.e., more selective

Wild type antibody can be used directly for conjugation

The robustness of required chemical steps: disulfide bond reduction and conjugation

with maleimide also makes this a very convenient approach for generating ADCs from

microgram scale for discovery research through kilogram scale necessary to support the

marketing of an approved agent

Maleimides react with thiols very quickly at neutral pH and with high selectivity over

amines, enabling well-defined conjugation

Maleimide linker payload are stable for months to years as stock solutions in appropriate

solvent and their rate of hydrolysis in aqueous solutions is negligible

Two step reaction scheme The interchain disulfide bonds are partially reduced with a reducing agent such as tris(carboxyethyl)

phosphine (TCEP), Dithiothreitol (DTT), 3-(diphenylphosphino) propionic acid etc.

The resulting free thiols are conjugated to a maleimide-containing linker-payload (MC-LP)

Procedure of Cysteine Mediated Conjugation

Challenges in cysteine mediated conjugation Many factors, including stoichiometry, pH, buffer concentration, temperature, mAb concentration,

co-solvent and its percentage affect cysteine mediated conjugation

In some cases, there is difficulty to identify the conjugation products due to positional isomers for the

conjugates with the same drug loading

Different mAb and linker payload show quite different behavior in conjugation

Hydrophobic linker-payloads may result in hydrophobic ADC that leads to rapid clearance in vivo

and aggregation issues

Quality Attributes for Conjugate Evaluation Drug Antibody Ratio (DAR): One of the most important quality attributes of an ADC is the average

number of drugs that are conjugated because this determines the amount of “payload” that can be

delivered to the tumor cell and can directly affect both safety and efficacy. Most investigators in the

ADC field have considered a drug loading range of 2–4 drugs/antibody as giving the optimal balance

between potency, pharmacokinetics and tolerability

Distribution of conjugates with different drug loading: This is an important ADC attribute

because different forms may have different pharmacokinetic and toxicological properties, the conjugates

with appropriate drug loading exert the optimum therapeutic effect

Unconjugated antibody (UmAb%): The competitive binding of naked mAb to antigen will block

the binding of ADC, and as a result, may cause the loss of therapeutic potency

Aggregation (Agg%): Aggregate levels in drug substance and final drug product are a key factor

when assessing quality attributes of the molecule, since aggregation might impact biological activity of

the biopharmaceutical; aggregated mAb will lose partially or totally its therapeutic properties or even

cause immunogenic reactions thus potentially further endangering patients' health

Residual drug: the quantity of free drug/degradants, poses concerns for differential toxicity and

potential safety issues. Residual amounts of drug or drug-related impurities may remain in the final

product as a result of incomplete removal by purification steps down-stream of the conjugation reaction

Goals

Develop reliable processes for ADC preparation of different scales

Investigate the kinetics of conjugation

Study the effect of stoichiometry of reducing reagent on DAR

Separation of conjugate species using HIC

Identification of these conjugate species

Materials

mAb-1 (10 mg/mL)

MC-LP (10 mM in DMSO)

VC-LP (10 mM in DMSO)

TCEP (10 mM in water)

Methods for characterization

UV spectrophotometry for protein concentration & Drug Antibody Ratio (DAR)

Hydrophobic interaction chromatography (HIC) for DAR, amount of unconjugated antibody

(UmAb%) and distribution of various loaded species (0, 2, 4, 6, 8)

Size exclusion chromatography (SEC) for amount of higher molecular mass species (Agg%)

Reversed phase chromatography (RP-HPLC) for DAR, amount of residual linker payload and related

species

Mass spectrometry for DAR, residual linker payload and related species and generally more in depth

characterization (peptide mapping, sequence variations, etc)

Goals, Materials, General Conditions and

Methods for Characterization

General conditions (previously explored)

pH: 6-8

Buffer: histidine, PBS

Temperature: 25-37°C

Co-solvent and percentage: 5-15% DMSO

min3 4 5 6 7 8 9

mAU

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*DAD1 E, Sig=280,16 Ref=360,100 (072815-HIC\072815HIC 2015-07-29 22-23-23\072815-1 1H.D) - DAD1 E, Sig=280,16 Ref=360,1

min3 4 5 6 7 8 9

mAU

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17.5

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*DAD1 E, Sig=280,16 Ref=360,100 (063015-HIC\063015HIC 2015-07-01 15-08-05\MAB-1.D) - DAD1 E, Sig=280,16 Ref=360,100 (06

min3 4 5 6 7 8 9

mAU

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min3 4 5 6 7 8 9

mAU

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min3 4 5 6 7 8 9

mAU

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*DAD1 E, Sig=280,16 Ref=360,100 (072915-HIC\072915-1HIC 2015-07-30 13-36-09\072815-1 20H.D) - DAD1 E, Sig=280,16 Ref=36

min3 4 5 6 7 8 9

mAU

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250

*DAD1 E, Sig=280,16 Ref=360,100 (072915-HIC\072915HIC 2015-07-30 09-12-13\072815-1 10H.D) - DAD1 E, Sig=280,16 Ref=360,

mAb-1

1 h

Effect of Reaction Time on the Conjugation Step

Conditions: mAb-1 and TCEP (3.2 eq), 2 h at RT; MC-LP (9.6 eq), 6.5% DMSO, RT

In the two step procedure for cysteine mediated conjugation, reduction is usually considered to be the

key step which finally controls the drug loading. As reported in literature, conjugation of the cysteine

thiols and the maleimide group is very facile, a fully completed reaction can be reached within 1h at 0°C

In our case, the conjugation of mAb-1 and MC-LP requires 10 h

Completed conjugation observed at 10 h

2 h

5 h

10 h

20 h

min4 6 8 10 12

mAU

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*DAD1 E, Sig=280,16 Ref=360,100 (080615-HIC\080615HIC 2015-08-06 18-51-43\080515-1.D) - DAD1 E, Sig=280,16 Ref=360,100

min4 6 8 10 12

mAU

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mAb-1+ VC-LP easy to identify the conjugates with different drug loading

mAb-1+ MC-LP More complicated conjugates

Standard HIC Chromatogram of Cysteine Mediated

Conjugation

Conjugation of mAb-1 and VC-LP gave an ADC mixtures in which conjugates with

different drug loading is easily differentiated in HIC chromatogram

The conjugates from mAb-1 and MC-LP show a complicated chromatogram, which are

difficult to identify for their drug loading

0 drug

2 drug 4 drug

6 drug

8 drug

0 drug

2 drug 4 drug difficult to identify the drug

loading of conjugates

Positional Isomers from Cysteine Mediated

Conjugation

Positional isomers from cysteine mediated conjugation cause additional complexity of

conjugate mixture

min3 4 5 6 7 8 9

mAU

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Crude conjugate

min3 4 5 6 7 8 9

mAU

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6

*DAD1 E, Sig=280,16 Ref=360,100 (073015-HIC\073015HIC 2015-07-31 19-44-46\072815-1 2-2.D) - DAD1 E, Sig=280,16 Ref=360,

min3 4 5 6 7 8 9

mAU

0

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4

6

8

10

*DAD1 E, Sig=280,16 Ref=360,100 (073015-HIC\073015HIC 2015-07-31 14-57-50\072815-1 P2.D) - DAD1 E, Sig=280,16 Ref=360,1

min3 4 5 6 7 8 9

mAU

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7.5

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min3 4 5 6 7 8 9

mAU

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min3 4 5 6 7 8 9

mAU

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Separation of Conjugate Isomers by Preparative

Chromatography (HIC)

Several conjugate isomers were separated by preparative HIC

Their drug loading was measured by UV absorbance and all peaks in crude mixtures were identified

based on drug loading

Those conjugates with higher drug loading (6 and 8) were not retrieved from column due to their higher

hydrophobicity

Loading = 0 2 4 6 8

Fraction 1

Fraction 2

Fraction 3

Fraction 4

Fraction 5

Fraction 6

min3 4 5 6 7 8 9

mAU

0

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5

7.5

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12.5

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17.5

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*DAD1 E, Sig=280,16 Ref=360,100 (063015-HIC\063015HIC 2015-07-01 15-08-05\MAB-1.D) - DAD1 E, Sig=280,16 Ref=360,100 (06

min3 4 5 6 7 8 9

mAU

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*DAD1 E, Sig=280,16 Ref=360,100 (072115-2D-HIC\072115HIC-2D 2015-07-24 08-55-02\072115-2.D) - DAD1 E, Sig=280,16 Ref=36

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TCEP Stoichiometry Study Targeting Optimum DAR

ADCs with specific DAR can be easily obtained by partial reduction of the native disulfides using

TCEP as reducing reagent. The level of drug loading in the final ADC is controlled at the reduction

step by modulating the molar equivalents of reductant added to the antibody

TCEP stoichiometry study indicated that in the conjugation of mAb-1 and MC-LP, using about 3.2

eq of TCEP resulted in the ADCs with DAR=4

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DAR

TCEP (eq)

Optimal TCEP Stiochiometry for DAR=4

TCEP (eq) 1 2 3 4 6

DAR 1.52 2.65 3.79 4.65 6.02

mAb-1

TCEP 1eq

TCEP 2eq

TCEP 3eq

TCEP 4eq

TCEP 6eq

min3 4 5 6 7 8 9

mAU

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min3 4 5 6 7 8 9

mAU

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mAU

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mAU

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0.2 mg, 20 uL

1 mg, 100 uL

Conjugation at Different Scales

Conditions: mAb-1 (10mg/mL, 20 mM histidine, pH=6) and TCEP (3.2 eq), RT, 2 h;

MC-LP (9.6 eq), 5% DMSO, RT, 15 h

5 g, 500 mL

10 mg, 1 mL

100 mg, 10 mL

1 g, 100 mL

25 g, 2500 mL

Conjugates Profile at Different Scale

Scale Volume DAR Agg% UmAb% Residual

drug%

0.2 mg 20 uL 4.06 1.79 4.57 <LOQ*

1 mg 100 uL 4.05 2.01 4.38 <LOQ

10 mg 1 mL 4.10 1.86 4.44 <LOQ

100 mg 10 mL 4.04 1.99 4.73 <LOQ

1 g 100 mL 4.02 2.05 4.92 <LOQ

5 g 500 mL 4.04 2.08 4.69 <LOQ

25 g 2500 mL 4.05 1.92 4.75 <LOQ

Conditions: mAb-1 (10mg/mL, 20 mM histidine, pH=6) and TCEP (3.2 eq), RT, 2 h;

MC-LP (9.6 eq), 5% DMSO, RT, 15 h

The excess of free drug is removed by buffer exchange using the following three ways:

NAP column (0.1~3 mL); dialysis (casettes, 1~50 mL); ultrafiltration/diafiltration

(50~2000 mL)

Conjugation of mAb-1 and MC-LP repeated very well at different scale

* Limit of Quantitation

Quality Attributes of ADC at Various Scales

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1 10 100 1000 5000 25000

DAR %Agg %UmAb

Scale of Reaction (mg)

Conjugation Reactors for Different Scale

300mL insert

Conjugation: 10~150mL Insert used in HPLC

vial

2mL HPLC vial

Conjugation: 100~500mL

8~25mL vial

Conjugation: 0.5~5mL

25~150mL reactor


500~5000mL reactor


Summary

A practical process was developed in mAbChem for cysteine mediated conjugation of

mAb-1 after thoroughly investigating the parameters involved in the reaction: pH,

stoichiometry, temperature, reaction time, buffer type, mAb concentration, co-solvent and

percentage

An optimal conjugate was obtained with ideal profile: DAR = 4; Agg% = 2; UmAb%=5,

Residual drug% < 0.1

The protocol was highly reproducible in our lab to give a consistent result at the scale

range of 0.2 mg~25 g

Longer conjugation time (10 h) may be needed for the conjugation of some specific

combination of antibody and linker payload, unlike the typical conjugation reported in

literature (0.5~2 h)

Conjugates with different drug loading were also separated as individual species from the

crude mixture by using hydrophobic interaction chromatography (HIC)

The drug loading of single positional isomers were successfully determined by UV

measurement

mAbChem poster-2015 ADC meeting

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