Development and Qualification of Analytical Methods to Support Low Concentration Drug Product in-use Studies.

The emergence of highly potent therapeutics with low expected clinical doses creates a challenge for analytical characterization of simulated drug product in-use samples. The low expected protein concentration (often µg/mL) and highly charged and sub-optimal sample matrices like 0.9% saline or 5% dextrose make ensuring dose solution stability and characterizing product quality changes difficult. Health authority expectations require analysis of low concentration in-use samples to be completed with suitable assays to ensure little to no changes are occurring during drug product dose preparation and administration, thus ensuring patient safety. However, characterization of these samples for protein concentration, size variants, charge variants and potency often necessitates additional analytical method development to improve sensitivity and compatibility with in-use samples. Here we report the development and qualification of reliable in-use methods to characterize simulated in-use samples to assist during drug product development.

Function-structure approach reveals novel insights on the interplay of Immunoglobulin G 1 proteoforms and Fc gamma receptor IIa allotypes.

Human Fc gamma receptor IIa (FcγRIIa) or CD32a has two major allotypes with a single amino acid difference at position 131 (histidine or arginine). Differences in FcγRIIa allotypes are known to impact immunological responses such as the clinical outcome of therapeutic monoclonal antibodies (mAbs). FcγRIIa is involved in antibody-dependent cellular phagocytosis (ADCP), which is an important contributor to the mechanism-of-action of mAbs by driving phagocytic clearance of cancer cells. Hence, understanding the impact of individual mAb proteoforms on the binding to FcγRIIa, and its different allotypes, is crucial for defining meaningful critical quality attributes (CQAs). Here, we report a function-structure based approach guided by novel FcγRIIa affinity chromatography-mass spectrometry (AC-MS) assays to assess individual IgG1 proteoforms. This allowed to unravel allotype-specific differences of IgG1 proteoforms on FcγRIIa binding. FcγRIIa AC-MS confirmed and refined structure-function relationships of IgG1 glycoform interactions. For example, the positive impact of afucosylation was higher than galactosylation for FcγRIIa Arg compared to FcγRIIa His. Moreover, we observed FcγRIIa allotype-opposing and IgG1 proteoform integrity-dependent differences in the binding response of stress-induced IgG1 proteoforms comprising asparagine 325 deamidation. The FcγRIIa-allotype dependent binding differences resolved by AC-MS were in line with functional ADCP-surrogate bioassay models. The molecular basis of the observed allotype specificity and proteoform selectivity upon asparagine 325 deamidation was elucidated using molecular dynamics. The observed differences were attributed to the contributions of an inter-molecular salt bridge between IgG1 and FcγRIIa Arg and the contribution of an intra-molecular hydrophobic pocket in IgG1. Our work highlights the unprecedented structural and functional resolution of AC-MS approaches along with predictive biological significance of observed affinity differences within relevant cell-based methods. This makes FcγRIIa AC-MS an invaluable tool to streamline the CQA assessment of therapeutic mAbs.

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function-Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants.

T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-of-concept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob-knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

Fc galactosylation follows consecutive reaction kinetics and enhances immunoglobulin G hexamerization for complement activation.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.

Initial results of the effects of diuresis on gadolinium enhancement in MR imaging of the abdomen.

RATIONALE AND OBJECTIVES: The authors performed this study to assess the effects of furosemide-induced diuresis on paramagnetic contrast material enhancement at magnetic resonance (MR) imaging of the kidney, liver, spleen, and psoas muscle. MATERIALS AND METHODS: Twenty-five patients (average age, 44.9 years; age range, 23-74 years; 13 men, 12 women) who were suspected of having unilateral renal hydronephrosis received 0.1 mmol/kg contrast material with a standardized injection and imaging protocol to assess organ signal intensity at 0-5 minutes after injection. All patients had a normal serum creatinine level. Imaging was performed with a 1.5-T magnet by using a fat-suppressed fast spoiled gradient-echo pulse sequence and a 70 degrees flip angle. Eight patients received 40 mg of furosemide 10 minutes before contrast material injection. RESULTS: The areas of the renal cortical and medullary signal intensity curves minus baseline in the unilateral normal kidneys were significantly greater in the group who received furosemide (P = .026 and P = .037, respectively). The areas of the renal cortical and medullary signal intensity minus baseline in the unilateral hydronephrotic kidneys were also significantly greater in the group that received furosemide (P = .036 and P = .026, respectively). There was a statistically significant increase in splenic enhancement (P = .02) and a tendency for increased liver (P = .09) and psoas muscle (P = .08) enhancement. CONCLUSION: Furosemide-induced diuresis appears to potentiate the cortical and medullary MR renogram, as well as the MR splenogram. A rapid shift in water compartmentalization from the intracellular to the extracellular space and increased renal water content with diuresis are possible explanations.

Gadolinium-enhanced T1-weighted renal and abdominal MR imaging: quantitative discrepancy between clinical and in vitro findings.

RATIONALE AND OBJECTIVES: This study was conducted to compare the magnetic resonance (MR) contrast medium enhancement of abdominal organs in vivo with the signal intensity (SI) values of known in vitro gadolinium solutions. MATERIALS AND METHODS: A phantom was imaged with the MR contrast medium gadodiamide (Omniscan; Nycomed, Princeton, NJ) of solutions at full-strength (0.5 mmol/mL), one-third, 1/10, and 1/100 concentrations. A fat-suppressed fast spoiled gradient-echo pulse sequence with flip angles ranging from 10 degrees to 170 degrees (at 20 degrees increments) was performed with a 1.5-T magnet. In 12 subjects, the SIs of abdominal organs were determined with identical imaging parameters, before and after administration of gadodiamide injection at 0.1 mmol/kg. RESULTS: As anticipated, the plot of SI in relation to gadodiamide concentration is nonlinear, with a decrease in SI due to T2 effects at concentrations above 0.05 mmol/mL. The kidney showed the highest SI after gadodiamide enhancement (125.2 +/- 11.6 [standard error] at 2.5 minutes), followed by the liver (76.5 +/- 11.5 at 1 minute) and spleen (57.26 +/- 9.35 at 30 seconds). The SI of the renal medulla (114.2 +/- 9.8 at 4.5 minutes) was approximately one-third that in phantom observations. CONCLUSION: The authors observed a marked discrepancy between empirical contrast medium performance in abdominal organs and SI values for comparable gadodiamide concentrations in vitro. One possible reason is the intracellular compartmentalization of water molecules in vivo. These results suggest a need for a better understanding of MR contrast medium performance in vivo.