Scale-up analysis for a CHO cell culture process in large-scale bioreactors.

Bioprocess scale-up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell-free mixing studies were performed in production scale 5,000-L bioreactors to evaluate scale-up issues. Using the current bioreactor configuration, the 5,000-L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5- and 20-L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000-L configuration can only support a maximum viable cell density of 7 x 10(6) cells mL(-1). Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000-L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing-related engineering parameters in the 5,000-L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed-batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000-L bioreactors may need optimizing to mitigate the risk of different performance upon process scale-up.

Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes.

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.

Identifying inhibitory threshold values of repressing metabolites in CHO cell culture using multivariate analysis methods.

Elevation of lactate, ammonia, osmolality, and carbon dioxide to inhibitory levels was reported to have adverse effects on cell growth and protein productivity in mammalian cell culture. Multivariate analysis methods were used to investigate the roles of these repressing metabolites in a fed-batch CHO cell culture for antibody fusion protein B1 (B1) production. Principal Factor Analysis methodology was applied to manufacturing-scale data of 112 cell culture runs, which identified threshold values of four repressing metabolites as follows: (1) ammonium levels above 5.1 mM inhibit cell growth; (2) both lactate and osmolality levels above 58 mM and 382 mOsm/kg affect cell viability; and (3) carbon dioxide levels at or above 111 mmHg reduce protein quality. These threshold values were then verified by simulations using Monod-type equations and Canonical Correlation. These results suggest that adverse effects on cell growth, productivity, and product quality may be minimized under the ideal cell culture condition, in which the peak values of all four repressing metabolites are maintained below the threshold values. This strategy was evaluated in 45 cell culture runs in 50-L bioreactors. Eight out of 45 runs were operated under the ideal condition, while the remaining 37 runs had at least one repressing metabolite with peak value at or above the threshold. In comparison to the remaining runs, the eight cell culture runs under the ideal condition had 17%, 40%, and 11% higher values in peak viable cell density, final B1 titer, and quality attribute, respectively. The unique methodology used in this study may be generally applicable in characterizing cell culture processes.

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study.

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.

Biomechanical comparison of stainless steel and titanium nails for fixation of simulated femoral fractures.

Flexible intramedullary nails are commonly used to treat femoral fractures in children. This study evaluated the biomechanical differences between stainless steel and titanium nails when securing transverse and comminuted fractures in a synthetic femur model. Retrograde flexible stainless steel and titanium nails placed in a divergent "C" pattern were mechanically tested, and axial rotation and compression stiffness were analyzed with a two-way ANOVA. Rotational stability was significantly greater for titanium nails than stainless steel nails for both fracture patterns. Axial compression stiffness was significantly greater for titanium nails than stainless steel nails for both fracture patterns. There was no statistical difference between materials for axial "failure" load that produced 5 mm of shortening. Titanium intramedullary nails were more stable than stainless steel nails in torsion and axial compression. Both materials stabilized simulated fractures at levels beyond physiologic non-weight-bearing loads without permanent deformation.

Biomechanical analysis of antegrade and retrograde flexible intramedullary nail fixation of pediatric femoral fractures using a synthetic bone model.

Biomechanical testing was performed to evaluate the stability of simulated transverse and comminuted femoral fractures after retrograde and antegrade flexible titanium intramedullary nail fixation. Ten synthetic adolescent-sized femoral bone models were used. Five underwent retrograde fixation with two C-shaped nails inserted from medial and lateral entry portals. The other five underwent antegrade fixation using one C- and one S-shaped nail through lateral entry holes just inferior to the greater trochanter. Retrograde nail fixation demonstrated significantly less axial range of motion and greater torsional stiffness than antegrade fixation in both transverse and comminuted fracture patterns. However, there appeared to be a biomechanical trend of greater resistance to shortening for antegrade nails.

Thoracoscopic multilevel anterior instrumented fusion in a goat model.

STUDY DESIGN: A survival study in a caprine model for evaluation of a thoracoscopic anterior scoliosis instrumentation construct for fusion. OBJECTIVE: To compare the efficacy of performing multilevel anterior thoracoscopic "scoliosis" fusion with both allograft and autograft bone. SUMMARY OF BACKGROUND DATA: The use of minimally invasive thoracoscopic spinal surgery has been increasing. Until recently, instrumentation methods for correcting scoliosis entirely with a minimally invasive approach have not been available. Although multilevel thoracoscopic instrumentation and fusion now is in use clinically at some centers, reports of its effectiveness in an animal model are limited. METHODS: A thoracoscopically implanted multilevel screw-rod instrumentation construct was used in a goat survival model, with fusion quality evaluated 16 weeks after surgery. Autogenous iliac crest bone graft was compared with an allogenic bone graft substitute. The quality of fusion was evaluated both radiographically and biomechanically. RESULTS: Thoracoscopic insertion of a multilevel screw-rod construct was demonstrated to be technically feasible. However, with regard to the quality of fusion, only 47% of the disc space "fusion ratings" with the use of autogenous iliac crest graft were considered fully fused by computed tomography scan evaluation. This compares with no ratings of full fusion in the allograft demineralized bone group and only a single rating of full disc space fusion in the control group, which received no bone graft. Torsional range of motion was the smallest in the group receiving iliac crest bone graft and the greatest in the control animals that did not undergo bone grafting. CONCLUSIONS: The lack of complete fusion at 4 months in the autogenous iliac crest group emphasizes the importance of the discectomy and bone grafting technique. The use of demineralized bone allograft as a sole source of interbody fusion is not supported.

EMG power spectra of intercollegiate athletes and anterior cruciate ligament injury risk in females.

PURPOSE: Females have a disproportionately high incidence of anterior cruciate ligament (ACL) injuries compared with males in analogous sports. Although the pathogenesis of this higher frequency has not been elucidated, gender differences in neuromuscular control of the knee may play an important role. This study evaluates EMG power spectra of the quadriceps and hamstring muscles during dynamic, fatiguing exercise to examine differences between male and female intercollegiate athletes. METHODS: Fifty-one collegiate basketball and soccer players (25 female, 26 male) were studied. Maximum voluntary contraction (MVC) was determined for knee flexion and extension. Three consecutive 2-min bouts of isokinetic knee flexion and extension exercise were performed at 40% MVC. EMG activity in the biceps femoris and vastus medialis obliquus was recorded using bipolar surface electrodes. RESULTS: MVC normalized to body weight was significantly greater in males than in females for the quadriceps (P< 0.01). Quadriceps coactivation ratios were significantly higher in females than in males during knee flexion exercises (P< 0.01). CONCLUSIONS: This study demonstrates differences in the EMG power spectra for females when compared with a matched group of males. Increased quadriceps coactivation in females may increase anterior tibial loads under dynamic conditions, thus placing the ACL at higher risk for injury in the female athlete.

Displaced pediatric supracondylar humerus fractures: biomechanical analysis of percutaneous pinning techniques.

Supracondylar humerus fractures are a common childhood occurrence. Displaced fractures are typically treated with closed reduction and percutaneous pinning. Controversy continues over the appropriateness of various pinning techniques. The most common include crossed or lateral pins. A biomechanical comparison of crossed pins, "parallel" lateral pins, and "divergent" lateral pins was performed using a pediatric synthetic bone model. Mechanical testing of each pin configuration was performed in extension, varus, valgus, internal rotation, and external rotation. The divergent configuration provided statistically greater stability than parallel pins under varus and valgus loading. Divergent pins had similar stability compared with crossed pins in extension, varus, and valgus testing. In axial rotation testing, crossed pins were more stable. If the surgeon feels confident in the ability of lateral pins to provide satisfactory fracture stability, divergent lateral pins provide greater stability than parallel lateral pins while avoiding ulnar nerve injury (associated with crossed pins).

Asymmetrical flexible tethering of spine growth in an immature bovine model.

STUDY DESIGN: A 12-week bovine survival study of tethering anterior spine growth that included untethered control subjects. OBJECTIVE: To determine the effects that a flexible cable attached to the anterolateral aspect of the thoracic spine has on spine growth in rapidly growing calves. SUMMARY OF BACKGROUND DATA: The search for a way to correct scoliosis without the use of an arthrodesis continues in an attempt to maintain normal spine mobility. Experience in the hemiepiphyseal stapling of long bones has provided a background rationale for attempting growth modulation in the spine. It is postulated that a mechanical tether to the anterior and lateral growth of the spine in a growing child with scoliosis may allow spontaneous correction of sagittal and coronal plane deformity obviating the need for an arthrodesis. METHODS: Eight calves (age, 3-4 weeks; weight, 47 +/- 4.6 kg) underwent right-side thoracotomies exposing the thoracic spine. Laterally directed anterior vertebral body screws were placed into each body and two vertebrae, either T6 and T7 or T8 and T9, were tethered with a stainless steel cable. After 12 weeks, radiographs were obtained to evaluate the degree of deformity that had developed. In addition, biomechanical testing to determine the range of motion in the tethered and untethered segments was performed. RESULTS: The calves increased their weight 153% during the 12-week postoperative period. The radiographic analysis demonstrated scoliosis of 11.6 degrees +/- 4.8 degrees in the tethered levels, as compared with 0.3 degrees +/- 1.7 degrees in the control segments (P < 0.0001). Similarly, kyphosis developed in the tethered segments (5.1 degrees +/- 5.8 degrees ), as compared with -1.8 degrees +/- 3.1 degrees at the control levels (P = 0.01). There was a significant wedging of the disc in the tethered (6.8 degrees +/- 1.6 degrees ) as compared with the untethered (0.7 degrees +/- 2 degrees ) segments (P < 0.0001). There was a trend toward lower height of the vertebrae on the tethered right side, as compared with the left side in the tethered segments (P = 0.075), whereas no side-to-side difference was noted in the control subjects (P = 0.48). Biomechanical analysis showed that the tether did not affect the range of motion in axial rotation or flexion-extension. However, the tether did restrict lateral bending as compared with that of the control subjects. When the tether was cut, the range of motion returned to levels matching that of the untethered control subjects. CONCLUSIONS: Anterolateral tethering of the spine creates kyphosis and scoliosis in this rapidly growing bovine model. The spinal tether limited motion primarily in lateral flexion. However, total lateral bending motion returned to levels comparable with control motion segments after removal of the tether. This method of spine growth modulation may provide a possible treatment for the correction of spine deformities without arthrodesis in patients who are skeletally immature. The exact mechanisms of growth modulation and the effects of tethering on disc function and integrity are unknown and deserve further study.

Endoscopically placed nasogastrojejunal feeding tubes: a safe route for enteral nutrition in patients with hepatic encephalopathy.

Patients with hepatic encephalopathy are at particular risk for aspiration when given oral or gastric feedings. An ideal strategy might combine distal enteral feeding with proximal gastric decompression, which is offered by a nasogastrojejunal (NGJ) feeding tube. One objective was to determine the efficacy and safety of endoscopically placed NGJ feeding tubes in patients with hepatic encephalopathy. Charts of patients who underwent NGJ tube placements between April 1997 and January 2000 were retrospectively reviewed. Two endoscopic techniques ("push" and "pull") were used. Eighteen patients (nine male and nine female) underwent 32 procedures. Twelve patients had undergone liver transplantation, four had decompensated cirrhosis, and two had fulminant hepatic failure. Twenty procedures used the push technique and 12 required the pull technique. The insertion time was shorter for the push technique compared with the pull technique (21.8 vs 39.6 min, P < 0.05). Enteral feedings were begun at an average of 5.2 hours after tube placement. The tubes remained in place for an average of 13.9 days. Complications related to the NGJ tubes included self-removal in eight, tube clogging in five, proximal migration in four, and intraduodenal migration of the gastric port in one. No aspiration episodes occurred. We conclude that NGJ feeding tubes may be placed endoscopically as a bedside procedure for patients with hepatic encephalopathy and provide a safe, efficacious, and rapid route for enteral nutrition in these patients.