Gayathri Ratnaswamy

Characterization of cysteine-linked conjugation profiles of immunoglobulin G1 and immunoglobulin G2 antibody-drug conjugates.

Two US FDA-approved antibody-drug conjugates (ADCs; Kadcyla(®) and Adcetris(®) ) have accelerated clinical interest in the potential of targeted cancer therapeutics as the next generation of oncology drugs that are designed to increase efficacy while reducing overall toxicity. Thiol conjugates are produced by partial reduction of the interchain disulfides, followed by conjugation with a drug-linker, resulting in a heterogeneous mix of molecules that differ with respect to the site of conjugation and the number of drugs per antibody. ADCs that have been characterized in this class have an immunoglobulin G1 (IgG1) framework and there is little information available on IgG2 ADCs. As IgG1s and IgG2s differ in the number of disulfides and molecular conformations, each subclass could lead to unique combinations of possible conjugation sites. We conducted in-depth characterization of two ADCs, an IgG1 and an IgG2 conjugated to monomethyl auristatin E. The results demonstrate that the IgG1 monoclonal antibodies favor conjugation to the cysteines between the light and heavy chains, whereas IgG2s demonstrate preference for the hinge region cysteines. The drug-loading distribution and conjugation sites of ADCs have been reported to influence pharmacokinetics, toxicity, and clearance. Therefore, an understanding of the conjugation profiles is important for the selection and engineering of ADCs.

Multivariate Analysis of the Sequence Dependence of Asparagine Deamidation Rates in Peptides

PurposeTo develop a quantitative scheme to describe and predict asparagine deamidation in polypeptides using chemometric models employing reduced physicochemical property scales of amino acids.MethodsDeamidation rates for 306 pentapeptides, Gly-(n−1)-Asn-(n+1)-Gly, with the residues n−1 and n+1 varying over the naturally occurring amino acids, were obtained from literature. A multivariate regression technique, called projection to latent structures (PLS), was used to establish mathematical relationships between the physicochemical properties and the deamidation half-lives of the amino acid sequences. Three reduced physicochemical property scales, amide hydrogen exchange rates (to describe the relative acidity of the amide protons) and flexibility parameters for the sequences were evaluated for their predictive capacity.ResultsThe most effective descriptors of the deamidation half-lives were reduced-property parameters for amino acids called zz-scores. The PLS models with the reduced property scales, combined with the hydrogen exchange rates and/or flexibility parameters, explained more than 95% of the sequence-dependent variation in the deamidation half-lives. The amide hydrogen exchange rate (i.e., amide proton acidity), hydrophilicity, polarizability, and size of amino acids in position n+1 were found to be the principal factors governing the rate of deamidation. The effect of amino acids in position n−1 was found to be negligible.ConclusionsChemometric analysis employing reduced physicochemical parameters can provide an accurate prediction of chemical instability in peptides and proteins. The relative importance of these various factors could also be determined.

Degradation products analysis of an Fc fusion protein using LC/MS methods

The following analytical methods have been used to identify and quantify degradation products in an E. coli expressed human immunoglobulin G Fc fusion protein in both liquid and lyophilized forms: two-dimensional AEX/RP/MS, limited proteolysis followed by LC/MS, and tryptic digestion followed by LC/MS/MS. After aging in a potassium phosphate pH 7.0 buffer for 3 months at 29 degrees C, peptide map analysis revealed that asparagine N78 (N297 according to Edelman sequencing) of the CH2 domain was the most rapidly deamidated site in the molecule probably due to the lack of the N-linked glycan on this asparagine, but this deamidation can be prevented under properly formulated conditions. This is the first report on the rate of deamidation on N297 of an IgG molecule without glycosylation. The active protein portion of the Fc fusion protein contains two methionine residues that are potentially susceptible to oxidation. Limited proteolysis was employed to cleave the active protein portion and measure the amount of oxidation. LC/MS analysis identified that the liquid sample aged at 29 degrees C for 3 months produced 40% oxidation, while the control sample contained only 4% oxidation on the active protein. In contrast to the aged liquid sample, the aged lyophilized sample showed no increase of deamidation or oxidation after storage at 37 degrees C for 8 months.

A Case Study of Nondelamination Glass Dissolution Resulting in Visible Particles: Implications for Neutral pH Formulations

Visible particles were unexpectedly observed in a neutral-pH placebo formulation stored in glass vials but were not observed in the same formulation composition that contained protein. The particles were identified as silica gel (SiO2 ) and polysorbate 20, suggesting dissolution of the glass vial. Time course studies were performed to assess the effect of variables such as pH, excipients, storage temperature, and duration on particle formation. Data suggest that glass dissolution occurred during the storage in the liquid state, as shown by increased Si levels in solution. Upon freezing, the samples underwent freeze concentration and likely became supersaturated, which resulted in the appearance of visible silica particles upon thawing. The glass degradation described here is unique and differs from the more commonly reported delamination, defined by the presence of reflective, shard-like glass flakes in solution that are often termed lamellae. This case study underscores the importance of an early assessment (during formulation development) of potential incompatibility of the formulation with the primary container.

Improving Workflow Efficiencies in Protein Formulation Laboratories Using Visual Basic for Applications

The protein formulation group develops stable formulations for both clinical and marketed protein therapeutics; this involves a systematic search of a large variable space. Several studies are set up each exploring the effect of a few variables at a time on protein stability. The amount of data generated from each study is substantial and is compounded as more studies are carried out. The business process from the data perspective was mapped to identify the data flow between the sub-processes. A number of redundancies in data entry that resulted in duplication of effort on the part of the researcher were identified. A custom Microsoft Excel Add-In package, using Visual Basic for Applications, has been developed in-house. To increase productivity and efficiency, at every step in the process, namely, planning, preparation, and execution and evaluation of the studies, these macros and templates allow automatic flow of information through the sub-processes. This has significantly reduced manual entry of data and errors in transcription and thereby led to savings in time and increased speed, reliability, and accuracy. Another advantage of using the macros is that the data format is standardized, facilitating the sharing of data and transfer of projects between teams. Further, metadata available through the use of the macros were used creatively to provide additional tools for overall project tracking.