Identification and characterization of an octameric PEG-protein conjugate system for intravitreal long-acting delivery to the back of the eye



Whitney Shatz1, Philip E. Hass1, Nikhil Peer1, Maciej T. Paluch1, Craig Blanchette 1, Guanghui Han2, Wendy Sandoval2, Ashley Morando3, Kelly M. Loyet3, Vladimir Bantseev 4, Helen Booler 4, Susan Crowell 5, Amrita Kamath 5, Justin M. Scheer 1, Robert F. Kelley6


  1. Protein Chemistry, Genentech Inc., South San Francisco, California, United States of America
  2. Microchemistry, Proteomics and Lipidomics, Genentech Inc., South San Francisco, California, United States of America
  3.  Biochemical and Cellular Pharmacology, Genentech Inc., South San Francisco, California, United States of America
  4. Safety Assessment, Genentech Inc., South San Francisco, California, United States of America
  5. Pre-clinical and Translational Pharmacokinetics, Genentech Inc., South San Francisco, California, United States of America
  6. Drug Delivery, Genentech, South San Francisco, California, United States of America


Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.

CovalX Technology Used (Click each option to learn more)




CovalX has designed a detection system specifically for higher mass proteins and protein complexes. The HM1 and HM2 use an ion conversion detector which greatly increases the sensitivity of the mass spectrometer. Higher mass ions: incident ions collide with a conversion dynode array which creates smaller secondary ions. Then, the secondary ions are reaccelerated into a SEM and detected with higher sensitivity because of their higher velocity. The use of these detector systems for high mass protein ions helps to prevent detector saturation and allow for better sensitivity and profiling.

Experiments were performed by obtaining 12 μM thick tissue samples from the brains of adult male Wistar rats. The tissues were mounted onto conductive glass slides, frozen for an hour and washed. Following washing, the tissue sections were placed in a bath of cold acetone for 30 seconds, removed and a bath of cold 95% ethanol for another 30 seconds and finally, placed in chloroform for 1 minute. Samples were compared using different procedures (standard, HFIP, Leinweber, tween and H2O2) before 0.5 μL of the specific matrix was added using a micropipette or an automated sprayer.  Each sample preparation was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.

Standard sample preparation: chloroform and ethanol washes, mixed with matrix of 20 mg/ml sinapic acid in acetonitrile:0.1% TFA (&;3, v/v)

HFIP sample preparation: addition of 20 mg/ml sinapic acid in pure HFIP onto washed tissue and then addition of recrystallization solution (20 mg/mL sinapic acid in acetonitrile:0.1% TFA (7:3, v/v))

Leinweber sample preparation: placement of tissue samples onto a droplet of 20 mg/ml sinapic acid in 90% ethanol that contained 0.5 % Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether and 0.1% TFA. The droplet was allowed to dry and a droplet of sonicated sinapic acid suspended in xylene was placed on top of the tissue sample before being dried in a vacuum desiccator. Finally, matrix droplets were applied again using 20 mg/mL sinapic acid solutions in 90% ethanol and 50% acetonitrile.

Tween sample preparation: the tissues were washed and a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA with a low concentration of polyethylene glycol sorbitan monolaurate) was added and allowed to dry.

H2O2 sample preparation: tissues were covered in a 3% solution of H2O2 before being incubated in a saturated vapor pressure chamber for 30 minutes and then dried in a vacuum desiccator. Finally, the samples were prepared using a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA (7:3, v/v)).

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Categories : Publications, High-Mass MS