Statistical Modeling of the Drug Load Distribution on Trastuzumab Emtansine (Kadcyla), a Lysine-Linked Antibody Drug Conjugate

Abstract

Trastuzumab emtansine (Kadcyla) is a recently approved antibody-drug conjugate produced by attachment of the anti-tubulin drug, DM1, to lysine amines via the SMCC linker. The resulting product exhibits a drug load distribution from 0 to 8 drugs per antibody that can be quantified using mass spectrometry. Different statistical models were tested against the experimental data derived from samples produced during process characterization studies to determine best fit. The Poisson distribution gives the best correlation for samples manufactured using the target process conditions (yielding the target average drug to antibody ratio (DAR) of 3.5) as well as those produced under conditions that exceed the allowed manufacturing ranges and yield products with average DAR values that are significantly different from the target (i.e., ≤3.0 or ≥4.0). The Poisson distribution establishes a link between average DAR values and drug load distributions, implying that measurement and control of the former (i.e., via a simple UV spectrophotometric method) could be used to indirectly control the latter in trastuzumab emtansine.

Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

“…Upon evolving from CEX heart-cuts 7-1, incremental increases of 957 Da corresponding to the addition of DM1 + linker are observed. A drug load of 0-8 is shown and the average drug load (or DAR) is situated between 3 and 4 which is in accordance with literature [11]. These are important quality attributes since they provide insight in the payload that can be delivered to the tumor.…”

“…With 80-100 lysine and only 8 interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation this despite the similar overall level of drugs incorporated per antibody (between 0 and 8 with an average of 3-4) [6]. Chromatography and mass spectrometry are at the forefront in ADC analysis and critical characteristics such as drug-to-antibody ratio (DAR), drug distribution and conjugation sites can be extracted out of the generated data [7,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. 1.…”

“…This biopharmaceutical, marketed as Kadcyla, is being used since 2013 in the treatment of human epidermal growth factor receptor 2 (HER2) positive breast cancer [8,11,29]. It combines the anti-HER2 antibody trastuzumab (Herceptin) with the cytotoxic microtubule-inhibiting maytansine derivative, DM1 (Fig.…”

“…Because HER2 is highly differentially expressed on breast tumor cells compared with normal epithelial cells, HER2 represents an ideal target for ADC therapy. With a drug distribution of 0-8, a DAR of 3.5 and various lysine residues available for conjugation, thousands of ADC species can be generated which demands for the best in terms of separation power [6,11]. In that respect, two-dimensional liquid chromatography (2D-LC) hyphenated to MS represents a powerful tool [30,31].…”

Highly Potent, Anthracycline-based Antibody–Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

“…14). For the maytansine-and auristatin-based linkerpayloads mentioned above, which are quite hydrophobic, it has been empirically determined that the "sweet-spot" for the average DAR is between 3 and 4, which is also reflected by the DARs of the two commercial products, Adcetris and Kadcyla, which have been reported to be 4.0 and 3.5, respectively (15,16). However, it is important to note that even with an average DAR in this range, the ADC preparation consists of a mixture of molecules comprising individual components with DARs ranging from 0 to 8 (14), each behaving differently with respect to their pharmacokinetic, efficacy, and safety profiles (17).…”