General Information

NISTmAb Overview

The NISTmAb material is a recombinant humanized IgG1κ expressed in murine suspension culture. It is an »150 kDa homodimer of two identical light chains and two identical heavy chains linked through both inter- and intra-chain disulfide bonds. The protein has low abundance post-translational modifications including methionine oxidation, deamidation, and glycation. The molecule also has N-terminal pyroglutamination, C-terminal lysine clipping, and glycosylation of the heavy chains. These and other product quality attributes were extensively characterized in the ACS book series "State of the Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization" for the initial batch of material (interim material 8670), used as the Primary Sample (PS). Multiple bulk substance containers were homogenized to form a second batch (14HB batch) of material that was aliquoted into 1 L containers. A single 1 L container of 14HB-XXY is diluted 10-fold and aliquoted as RM 8671 lot 14HB-D-XXY.

A vial of RM 8671 contains 800 µL of 10 mg/mL IgG1κ monoclonal antibody in 12.5 mmol/L L-histidine, 12.5 mmol/L L-histidine HCl (pH 6.0). The material has undergone industry standard upstream and downstream purification to remove process related impurities.
Schematic 1. Humanized IgG1κ domains of NISTmAb are marked as variable (V) or constant (C) with subscripts for light (L) or heavy (H) chains. Distinct heavy chain domains are numbered 1, 2, or 3 starting from the N-terminus. Black lines indicate intra- and inter-disulfide bonds, with cysteine residues numbered as they appear in the light or heavy chain sequence starting from the N-terminus. The IdeS cleavage site is indicated with scissors. Post-translational modifications (PTMs) that are commonly identified in the NISTmAb are listed.

NISTmAb Technical Definitions

Reference Material 8671 (RM 8671) NISTmAb derived from multiple production lots (using the same process as PS 8670) that was homogenized and diluted as the publicly available reference material. Publicly available at 10 mg/mL in 800 uL aliquots.

Primary Sample 8670 (PS 8670) NISTmAb derived from a single production lot early in the product lifecycle and used during the initial development stage. This lot was reserved as the final in-house primary reference standard. Material is reserved for NIST in-house use and interlaboratory studies as needed (10 mg/mL and 100 mg/mL)

Reference Material Information Sheet (RMIS) Official NIST document describing material properties, NIST assigned values, intended use, and proper handling instructions.

Lifecycle management plan: NISTmAb material handling plan devised to follow industry standard practices and provide long-term availability of material with consistent product quality attributes. A brief figure summarizing this plan is below, while a detailed discussion can be found here .

Lifecycle management plan for NISTmAb RM 8671. Solid arrows represent the use of material to mix, vial, dilute, etc. Dashed gray arrow represents a value assignment exercise

NISTmAb F.A.Q.

The recommended storage temperature for NISTmAb RM 8671 is -80°C. If this is not possible, the maximum storage time under given conditions and for specific intended uses can be found in the RMIS.
Upon removal from -80°C, the vial of RM 8671 should be thawed at room temperature for approximately 30 minutes, or until no residual ice crystals remain. Ensure the solution is homogenous by gently inverting the vial five times. Briefly spin down the vial before use.
The background solvent used for formulation of NISTmAb is 25 mM L-Histidine HCl, pH 6.0. A detailed protocol to prepare the formulation buffer can be found here
NISTmAb RM 8671 is only available at the 10 mg/mL concentration.
The primary amino acid sequence can be found in the RMIS . For more information, click here .
The theoretical extinction coefficient (ε) of NISTmAb RM 8671 is 1.42 mL · mg-1 · cm-1. For more information related to this value, click here
The pI of NISTmAb RM 8671 is 9.18. For more information related to this value, click here