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Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

Innovations in the biomanufacturing industry are predominantly incremental in nature, but there are occasional major technological shifts that occur as existing technologies reach their limits and as additional constraints are imposed by market forces or regulatory bodies. These disruptive innovations tend to have a seismic effect, forcing companies to adopt novel strategies and business models in order to remain competitive. In this article, we discuss how technological innovation can address some of the current challenges in biopharmaceutical manufacturing by anticipating shifts in market dynamics and cost structures. We consider bioprocessing standards, unit operations, best practices, enabling technologies and look at potential entry points for innovation where technologies are likely to evolve incrementally and where more disruptive, radical changes may flourish, including quality by design and process analytical technology. We conclude that the biomanufacturing industry can only thrive in the changing market environment by embracing innovation and becoming more adaptable.

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2–0.5 log10). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log10). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log10). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 257–267, 2002.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'‐nuclease Product‐Enhanced Reverse Transcriptase (TM‐PERT) assay for measuring reverse transcriptase (RT) activity in cell‐culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory‐scale cell‐culture harvests (108−1013pU/mL) were substantially above the detection limit of the TM‐PERT assay (∼106pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory‐scale chromatographic runs, sufficient RT activity was present in mAb‐containing eluates to accurately calculate its log10reduction value (LRV), typically between 2 and 4 log10per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM‐PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to γδ T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for γδ T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain γδ T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing γδ T cells further extends the list of potential candidates for antigen presentation to γδ T cells.

Abstract: The major histocompatibility complex (MHC) of the BALB/c mouse contains three genes encoding classical class I molecules, as well as at least 32 nonclassical class I genes. Although much is known about the genes encoding the classical class I molecules, the majority of the nonclassical genes have not been characterized. This report describes a newly identified nonclassical class I gene,Thy19.4, which contains an open reading frame and resembles in several regards the genes encoding classical class I molecules. The similarities include shared amino acid sequence motifs which suggest that the putative Thy19.4 molecule may assume a tertiary structure similar to that of the classical class I molecules, as well as widespread transcription in a variety of tissues. However, unlike the classical class I genes, theThy19.4 gene maps approximately 1 cm distal to theTla region of the MHC, in the same region as the gene encoding theHmt element of the maternally transmitted antigen.

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2d, D3d, and D4d, in addition to H-2Ddand H-2Ld. We have determined the DNA sequence of the D2dgene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2dgene is similar to other class I genes. It also contains similar 5' regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2d molecule, we generated an exon-shuffled construct containing the promoter and exons 1–3, encoding the signal peptide, a1, and a2 external domains of the D2dgene linked to exons 4–8, encoding the a3, transmembrane and cytoplasmic domains, of the H-2Ddgene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the a3 domain of H-2Dd, as well as by other class I-specific mAbs. Although D2d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.